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https://f1000research.com/articles/8-1107/v1	16 Jul 19	{ "type": "Research Article", "title": "A controlled study on an instrument that couples active learning with technology: student video creation", "authors": [ "Gyzelle P.V. Nascimento", "Daniel C. Moreira", "Alexis F. Welker", "Daniel C. Moreira", "Alexis F. Welker" ], "abstract": "Background: Active learning strategies and the use of technology in classes have been widely indicated to enhance learning. Although much has been discussed on these topics, few studies have addressed them with adequate experimental designs. Therefore, this study investigated the effect of a strategy coupling active learning methodology and technology –video lectures production by students – on the students’ learning in comparison with traditional approaches. Methods: To investigate the impact of video production on students’ learning, approximately half of one class of undergraduate students in a Pharmacy program attended traditional classes on one of its modules, while the other half was instructed to elaborate video lectures about the same content. We recorded their scores in two exams on the topic covered by the video lectures, one prior to intervention and the second after the intervention. We also recorded their score in a final exam at the end of the course, which covered all modules in the course, and applied a questionnaire to assess students’ perceptions about the applied methodology. Results: The average score of the students in the video group became 46% higher than the control group’s score. The score on the final exam at the end of the course showed no difference between groups. Most of the students reported that the video lectures they produced in class improved their academic performance. Conclusions: The video lecture production activity, a teaching instrument that relies on active learning and technology, was able to improve learning indicators of a group of randomly selected students in comparison with a control group of students who attended traditional expository classes given by an instructor.", "keywords": [ "Learner-centered", "Pharmacognosy", "Pharmacy", "Satisfaction in studying", "Teaching strategy", "Video lecture" ], "content": "Introduction\n\nTeaching approaches that involve active learning lead to greater motivation to study and increased interaction among students in comparison to expository class given by a professor (Conway et al., 2010; Gleason et al., 2011). Other possible outcomes, such as an increase in learning, have not been unequivocally confirmed so far. Some studies have shown that active learning is no more effective than passive approaches in increasing the level of learning (Conway et al., 2010; Haidet et al., 2004), whereas other studies have shown that some active teaching strategies do increase the level of learning (Letassy et al., 2008). This discrepancy seems to depend on the context in which the method is applied and measured, as well as on the control groups. One factor that may explain such contradictory findings on the impact of active strategies on learning levels is the academic background of students (Haak et al., 2011). There are diverse active learning strategies (Gleason et al., 2011), such as team-based learning (Conway et al., 2010; Parmelee & Michaelsen, 2010), case-based learning (Tayem, 2013), learning by discovery (Spencer & Jordan, 1999), inquiry-based learning (Banchi & Bell, 2008), web-based study activities (Alonso et al., 2005), collaborative and cooperative learning (Smith & MacGregor, 1992), evidence-based teaching/learning (Kenyon et al., 2016), and problem-based learning (Barrows & Tamblyn, 1980; Hmelo-Silver, 2004; Kilroy, 2004). Many publications addressing active learning strategies focus on presenting their theoretical benefits, describing the methodology used, and making recommendations (Parmelee & Michaelsen, 2010; Passos et al., 2006); however, most of them do not show actual data to support them. Therefore, more studies are needed to fully understand the impact of active learning and teaching on students’ performance.\n\nMany publications have explored the use of new teaching technologies and methods (Cubas Rolim et al. 2017; Oliveira et al., 2010; Pandza & Masic 2013; Pertry et al., 2014; Sé et al., 2008). Technological tools can provide notable advantages, giving students flexibility in choosing when and where to study, and increasing their access to information (Fernandez et al., 2014; Kenny, 2002; Weed et al., 2014). Potentially beneficial technologies include: videos used to summarize information at the end of a class in place of a questionnaire or slide review (Sarikcioglu et al., 2011), distance education to promote active learning (Cravener, 1999), medical simulations to build student confidence in clinical practice (Keegan et al., 2012; Reilly & Spratt, 2007), and seminars to improve interactivity among students (Brunton et al, 2000). However, few studies with adequate control groups have investigated the impact of new educational technologies on levels of learning.\n\nBoth active learning and new teaching technologies may provide some benefits, but adequate assessments of their impact on learning levels are rare. Even less is known about the effect of combining active learning strategies with technological tools. To form a better view of the effect of active learning coupled to technological tools on learning, we instructed undergraduate students to produce video lectures and compared their academic scores with students that did not produce video lectures. In addition to measuring their academic performance in the course, we also assessed their degree of motivation and satisfaction derived from this learning approach using a questionnaire.\n\n\nMethods\n\nThis study involved 50 students attending a Pharmacognosy course during the fifth and sixth semesters of a Pharmacy program at the Integrated Faculties of the Educational Union of Planalto Central (UNICEPLAC, Brasília, Brazil). To be included in the study, the students had to be enrolled in the Pharmacognosy course as part of the Pharmacy graduate program at UNICEPLAC. Therefore, students enrolled in other programs or not enrolled in the Pharmacognosy course were excluded from the study. The study size was determined by the number of undergraduate students that met the inclusion criteria and agreed to participate in the study; all eligible students were recruited as follows. At the first class of the Pharmacognosy course, the students were informed about the intention to conduct the study on the use of active learning methodologies coupled with technological tools. At this moment, written informed consent was obtained. At the second class, students who were not present in the first class were informed, also agreed to participate in the study and signed the informed consent. This course covers several topics related to the use of active substances of plant and animal origin during one semester with a total workload of 54 hours. Three weeks after the beginning of the course, the professor applied a written test to evaluate the students’ previous knowledge about the use of herbal medicines, a topic often discussed in the media and covered in previous courses in the Pharmacy program.\n\nTo investigate the impact of video production on students’ learning, motivation, and satisfaction, students were randomly divided into two groups of equal size. At the fifth class, the students were randomized by placing each student who was arriving at the classroom alternately in one of the two experimental groups. Although no attempt was made to match cases and control, the two groups had similar demographic characteristics (p > 0.05; Table 1). Both groups studied the use of herbal medicines module for ten hours in separate rooms using different learning strategies. The control group continued to study in the way they had done previously, by listening to a four-hour lecture given by the professor using visual resources; in this group, students were encouraged to participate through questions and comments. The video (intervention) group was divided into seven teams with a maximum of five students that were instructed to produce a video lecture on the use of herbal medicines. These students worked together, without the supervision of any instructor, using books, online resources, cameras, cell phones, and computers to create their videos (see extended data for videos (Nascimento et al., 2019b)). As the use of herbal medicines module is equivalent to approximately 20% of the course content, students in the video group experienced 20% of the course using a different methodology from the control group.\n\n*This is the terminology used by The Brazilian Institute of Geography and Statistics.\n\nAfter studying the use of herbal medicines, all students were once again assessed on their knowledge of the field through a second written test. Then, the whole class watched the video lessons produced by students and filled out a questionnaire (extended data (Nascimento et al., 2019b)) on their preferred information sources and the degree of motivation and satisfaction they had experienced while studying using the two different learning methods. A third test (the final exam; see extended data (Nascimento et al., 2019b)) was applied at the end of the semester and evaluated all topics covered by the course. Both groups were evaluated by the same tests.\n\nThis research project was approved by the Research Ethics Committee registered through the Research Ethics Committees (CEPs, acronym in Portuguese) and the National Research Ethics Commission (CONEP, acronym in Portuguese) also known as the CEP/CONEP system (approval number 13930013.6.0000.5058/244,173). Written informed consent from all subjects involved was obtained for participation in the study and subsequent publication of the videos they produced.\n\nScores of groups before and after the intervention were compared using the Wilcoxon’s test. Differences between scores within each group were analyzed using the Mann-Whitney test. The homogeneity of the qualitative variables between the “Control” and “Video” groups was verified using Fisher's exact test. All analyses were performed using R software (version 3.0.1).\n\n\nResults\n\nStudents were evaluated twice on the use of herbal medicines in order to verify the extent to which producing their own video lectures affected their learning. In the first evaluation, before any learning method had been used, the students in the video group had an average score that was 13% lower than that of the control group (p < 0.05). After studying the use of herbal medicines in two different ways, the average score of the students in the video group had become 46% higher than the control group’s score (p < 0.001; Figure 1). The control group’s score decreased and the video group’s score increased between their first and second evaluations (p < 0.01). The final exam at the end of the course, which included all content covered during the semester, showed no difference between the scores of the video and control groups (p > 0.05; Figure 2). See underlying data for results for each student (Nascimento et al., 2019a).\n\nStudents in the video group answered fewer questions correctly than the control group on the first test, but more questions correctly on the second test. The asterisks denote significant differences between the first and the second exams within the same group (p < 0.01).\n\nn.s., nonsignificant.\n\nThe results obtained from the questionnaire show that students use online resources on the Internet as their main source of information (Figure 3). They habitually watch video lectures, and most of them believe that these videos improve academic performance (Table 2). When asked who or what factors had encouraged them to watch videos, most cited their professors. In order to investigate the factors influencing the students’ perception that video lessons improve learning, students were asked to rate various factors. None of the suggested factors were perceived as responsible for their academic performance improvement (Table 3). When asked which learning methodology they preferred, most preferred the traditional approach, which they were used to (Table 2). Most of the students (92 %) in the video group reported that producing video lectures during class as a studying strategy stimulates learning when compared to only watching them (Figure 4).\n\n\nDiscussion\n\nIn this study, we investigated the impact of a new teaching instrument that combines active learning with the use of videos by enabling students to create their own video lectures. We observed that levels of knowledge on the topic covered in the videos rose in the group of students who produced videos, in comparison with those who attended expository classes given by an instructor. This finding was reinforced by the opinion of the students, who believed that this instrument improved their performance. This research also revealed students’ great attraction to the Internet and related technologies. Most students believe that simply watching video lessons (not produced by themselves) stimulate and improve learning. Interestingly, the greatest motivator for the use of video lectures mentioned by the students was their own instructors.\n\nThe observed increased learning supports the idea that active learning can be more beneficial than relatively passive learning methods. Other studies measuring learning have also demonstrated that active learning methods can increase knowledge (Wiecha et al., 2006). However, other studies using adequate control groups have not found any learning differences between active and more passive learning methods (Haidet et al., 2004). One factor that may explain this discrepancy is the students’ own educational background. Active learning has been shown to provide more benefits for students with weaker academic background than for those with more knowledge (Haak et al., 2011). The students in our research project were enrolled in a private institution, which in Brazil is expected to have students with lower scores in pre-university academic exams than those in enrolled in public universities. Their marked increase in learning through the creation of videos shows that they were well suited to benefit from this active learning instrument. Nevertheless, our results do not allow us to make the generalization that active learning always leads to greater learning gains for every group of people.\n\nThere is evidence that, for some groups, active learning methods are no more efficient than more passive methods. One reason is that the traditional approach (expository lectures) varies greatly from professor to professor. Instructors who are very skilled in establishing empathy, creating interest, and offering very popular classes might achieve better learning indicators from the students than those who do not have such abilities (Francis & Tannuri-Pianto, 2012; Sampaio et al., 2011). Indeed, improving the quality of teacher-student interaction causes a sharp increase in student learning (Allen et al., 2011). Even with the successful implementation of a more active system of learning, low professor availability causes great dissatisfaction among students (Gahutu, 2010). It is therefore assumed that students of instructors who are experts in engaging activities, such as public speaking and story-telling (Latey, 2000; Leach, 2005; Stein, 2009) learn more than the students of professors who do not teach in this way. This could be one explanation for the low learning rates associated with purely lecture-based teaching observed in some studies (Cook et al., 2008).\n\nOur student participants reported that the Internet was the learning instrument they used the most. This reflects the increasing use of technology (Chaudhry et al., 2010; Erickson et al., 2010; Giordano & Giordano, 2011). However, our students’ high reliance on the Internet may also indicate that they could be less well educated than some others. The Internet might provide incorrect or false information (Barrie & Presti, 1996; Fraval et al., 2012), including many videos that contain misleading or improper content (Murugiah et al., 2011; Sood et al., 2011; Tourinho et al., 2012). Our results showed that the student participants used books less often than they used the Internet, which may indicate some deterioration in the quality of their learning. Specialized health professionals, even with ample access to the Internet, still use books as their main source of information for solving medical problems (AlGhamdi, 2009). On the other hand, most (59%) of highly qualified European doctors, 99% of whom have regular Internet access, report using article research databases like PubMed to access scientific content (Kritz et al., 2013). Considering the reliability problems of Internet data and the dominance of advertising (Hossler & Conroy, 2008; Jenkin et al., 2014; Vance et al., 2009), it is important to assess the causes and outcomes of our students’ tendency to avoid books.\n\nDespite many problems with Internet use, there are some obvious advantages, such as the availability of information. However, statements about the potential benefits of using the Internet or new technologies are often unsubstantiated by data and based instead on opinions (Carlon et al., 2012; Pandza & Masic, 2013; Weed et al., 2014). Most studies have focused on factors such as satisfaction rather than on learning (Curran & Fleet, 2005; Kerfoot et al., 2006; Narula et al., 2012). Some studies have shown that the Internet has a positive impact on learning, but many of these had no control group or an inadequate control group (Karpa, 2012; Oliveira et al., 2010; Pertry et al., 2014; Rangel et al., 2010). This makes it impossible to identify the most efficient form of teaching/studying for increased learning (Cook, 2009; Kleinpell et al., 2011; Lantz, 2010; Steele et al., 2009). In contrast to expectations, comparative studies with an adequate control group, such as lectures given by highly skilled instructors, often reveal that there are no differences between technology-based and traditional learning methods (Chao et al., 2012). We would expect any form of teaching to provide benefits when compared with the absence of a stimulus (Cook, 2009; Lipscomb et al., 2009). Indeed, we found that producing video lectures enhance learning on a specific topic (the use of herbal medicines) of a Pharmacognosy course. Additionally, the present study showed that most students (from both groups) believe that watching video lectures improve their academic performance and those who had produced video lectures (video group) believed that producing video lectures stimulates learning.\n\nIn conclusion, this study shows that a new teaching instrument that combines active learning with the use of technology through the creation of student-designed video lectures was able to increase the learning of a group of randomly selected students in comparison with a control group of students who attended traditional expository classes given by an instructor. This result corroborates many articles that have theorized that active learning is better than passive learning, and that using videos and other new technologies can increase learning. To date, few studies with adequate control groups have been able to confirm these theories.\n\n\nData availability\n\nfigshare: Nascimento et al. Student video creation Dataset 1 CC0. https://doi.org/10.6084/m9.figshare.8343998 (Nascimento et al., 2019a)\n\nThis project contains the following underlying data:\n\nNascimento et al. student video creation Dataset 1.xlsx (Students’ responses to the questionnaire and exams scores)\n\nfigshare: Nascimento et al. Student video creation Extended data. https://doi.org/10.6084/m9.figshare.8345618 (Nascimento et al., 2019b)\n\nThis project contains the following extended data:\n\nVideo 1.mp4 (video lecture produced by students)\n\nVideo 2.mp4 (video lecture produced by students)\n\nVideo 3.mp4 (video lecture produced by students)\n\nVideo 4.mp4 (video lecture produced by students)\n\nVideo 5.mp4 (video lecture produced by students)\n\nVideo 6.mp4 (video lecture produced by students)\n\nVideo 7.mp4 (video lecture produced by students)\n\nNascimento et al. student video creation first exam.pdf (first test about the use of herbal medicines)\n\nNascimento et al. student video creation second exam.pdf (second test about the use of herbal medicines)\n\nNascimento et al. student video creation final exam.pdf (pharmacognosy final exam)\n\nNascimento et al. student video creation questionnaire.pdf (questionnaire)\n\nNascimento et al. Student video creation avaliação 1.pdf (first exam, original Brazilian Portuguese version)\n\nNascimento et al. Student video creation avaliação 2.pdf (second exam, original Brazilian Portuguese version)\n\nNascimento et al. Student video creation avaliação 3.pdf (pharmacognosy exam, original Brazilian Portuguese version)\n\nNascimento et al. Student video creation questionário.pdf (questionnaire, original Brazilian Portuguese version)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors thank Robertta P. 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Choe", "expertise": [ "Reviewer Expertise Life Science Education", "Evidence-Based Teaching", "Assessment" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper highlights an important issue in online instruction: active learning and technology and how they can be merged to fit into the current learning environment. As the authors state, there is a need for further investigation of this intersection. This paper attempts to fill in the gaps in this space. The video production intervention provides an example of how instructors can incorporate meaningful active learning into their classrooms. The results showing improved learning outcomes for the intervention group is promising and aligns with much of the literature supporting student-centered learning.\n\nIs the work clearly and accurately presented and does it cite the current literature?\nThe study is presented clearly and accurately. However, the title may be misleading because technology may not be the only independent factor in this experiment. The learning objective of the intervention and control arms differ on Bloom’s taxonomy of learning. The video production intervention is higher on Bloom’s taxonomy than the undirected self-study in the control group. Creation, as well as the other higher learning objectives on this taxonomy, has been linked to improved learning outcomes. This possibly confounds the intervention because the higher order learning objective may be responsible for the learning gains, not necessarily the engagement of technology. If the students in the intervention arm were asked to produce something on paper instead of video, Bloom’s taxonomy would suggest that similar learning gains would likely result.\n\nThe manuscript cites many relevant studies but it would be strengthened by including more recent studies that address some of the gaps that the authors mention. For example: “This result corroborates many articles that have theorized that active learning is better than passive learning, and that using videos and other new technologies can increase learning. To date, few studies with adequate control groups have been able to confirm these theories.” Reference to Mayer’s multimedia learning principles could fill in part of the aforementioned gap. Richard Mayer is a cognitive psychologist at the University of California, Santa Barbara and whose research focuses on learning outcomes in multimedia learning with controlled psychological testing environments. The manuscript could be improved by including the findings on Mayer’s handbook on Multimedia Learning.\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\n\nThe statistical analysis and its interpretation are appropriate, but the sample size is small. Increasing the sample size is critical for improving the statistical power of this test. Effect size comparisons for each group could be added because the sample data is paired.\nAre the conclusions drawn adequately supported by the results?\nThe conclusions are drawn adequately but additional clarification is required. The control group shows a high first exam score followed by decreased second exam score, while the intervention group shows a lower first exam score followed by an increased second exam score. This finding possibly aligns with the findings of a 2006 study by Roediger and Karpicke, Test-Enhanced Learning. Could the authors consider this paper and its implications in the discussion?\n\nFigure 3 indicates an important shift in the learner’s experience and the increasing use of the internet for study. This is an area that is important for discussion and further investigation. The questionnaire data revealed that a majority of the students were using the internet as a studying resource and that they believed watching the videos were beneficial for their learning. But it seems that students are also aware that more complex learning objectives can increase learning outcomes. Looking at the dataset, it seems that many students from the control group also indicated yes to the question shown in Figure 4. Yet, Fig. 4 only shows the responses from the students in the video group. Could the authors please clarify why the control group responses were not included here?\n\nCould the authors please provide more information on the instructions given to the video production group?\n\nCould the authors please provide more information about the four-hour lectures given by the professor using visual resources? Did the video production group also listen to the lecture? What were the visual resources? Did both groups receive these visual resources?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "73943", "date": "12 Nov 2020", "name": "Samuel A. Tisherman", "expertise": [ "Reviewer Expertise Resuscitation", "simulation", "surgical skill performance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGiven that previous studies regarding active learning and new educational technologies have not been definitive, this study seeks to assess the effects of active learning approaches and new teaching technology on student learning. The authors randomized students in a pharmacy course to an active learning activity utilizing technology (creation of lecture videos) vs. traditional, more passive receipt of lecture materials for one module focused on herbal medications within the course on pharmacognosy. Learning was assessed with a pre-test and a post-test on herbal medications, and a final exam on the whole course, only 20% of which was on herbal medication. Students who engaged in the active learning activity with technology outperformed those who passively learned on the post-test focused on herbal medications. However, there was no difference in performance on the final exam for the course as a whole. The authors suggest that their result corroborates previous studies demonstrating benefits of active learning and using videos or other new technologies.\nIntroduction: The authors review various forms of active learning and technological tools. They note that prior studies have arrived at disparate conclusions regarding the efficacy of active learning paradigms vs. passive learning and note that the context of the method, the control groups, and the background of the students may mediate the effect.\nAlthough the hypothesis was presumably that the combination of active learning and use of technological tools would improve performance, this is not specifically stated.\nMethod: Enrollment of the students is not clear. If they were asked to sign consent for participation, were there any students who did not consent? If so, did they not participate in either learning activity?\nThe methodology does not actually allow one to draw any conclusion separately about the use of active learning vs. the use of technology. For instance, it is possible that the results would have been the same if both groups prepared a lecture, but only one group used video technology. If both variables (active vs. passive learning, technology vs. no technology) are to be addressed, a 2X2 design would be preferable.\nThe authors noted that each group studied the module for 10 hours in separate rooms using different learning strategies. Did this include the lecture and study time in the control group and the time for background research and video development in the intervention group? Over how many days were the 10 hours distributed? The control group received a four-hour lecture. How was the lecture delivered? Was it a single four-hour lecture, four one-hour lectures, 8 30-minute lectures? Were the students merely instructed to watch it at some point during the 10 hours? Was there an instructor present for the hours of study outside of the lecture?\nThe intervention group was divided into teams of five students that were instructed to produce a video lecture on the use of herbal medicines. Were they limited to 10 hours of work as well? Why was an instructor not present for assistance? This brings in a third variable that may have affected the learning process: 1) active vs. passive learning, 2) technology vs. no technology, 3) instructor present vs. no instructor present.\nHave the written tests been previously validated or used for publication? Were these tests standard for this course? If not, this should be noted as a limitation.\nThe examinations have open-ended questions. Who graded the examinations and was this person (or persons) blinded to student group assignment?\nHow much of the final exam included the topic of interest (the use of herbal medicines)? Was the content of the exam related to herbal medicine equivalent to the 20% of the course? How much time had passed from the educational intervention to the written exam? This information would be critical for evaluating the success of the intervention.\nCan the authors describe why they picked their list of possible reasons for the learning improvement associated with video lectures? Can they define the reasons, e.g. what does Fixation mean? Is there evidence that these are the most important reasons? If so, please cite.\nResults: In the Introduction, the authors note that the background of students may influence whether or not active leaning paradigms are successful as compared to more passive learning. Unfortunately, we learn relatively little about important background aspects of the students other than their basic demographics. What is their prior experience with producing lectures, with employing video technology, etc.? Do the learners receive any instruction on how to use the internet for educational purposes? Does the curriculum include other self-study components?\nRegarding the survey on preferred sources of information as reported by students, it is uncertain whether or not the students classified online textbooks and articles as “internet” or “books”/”articles”. To give some context, are these students assigned video lectures in most of their classes? Are the video lectures supplementary or the primary mode for transferring information?\nWere the student-developed video lectures critiqued or otherwise evaluated by the other students or the faculty?\nHow did the learners do on the subset of the final exam that was focused on the use of herbal medicines? This would be an important result to assess retention in the active learning group. There is no a priori reason to believe that the video lecture group would do better than the control group on other parts of the course for which they all received the same education.\nDiscussion: The authors observed that knowledge improved in the group of students who produced videos in comparison with those who only received instructor-led videos. In addition, the students believed that this experience improved their performance. It is curious then that most students believe that producing video lectures stimulates learning but none of the surveyed reasons were endorsed. Can the authors comment on this?\nFollowing the intervention, 50% of the students still preferred traditional classes vs 40% that preferred producing video lessons (Table 2). Were the results similar in the video group alone? It would be surprising since 92% of them thought that producing video lectures stimulated learning when compared to only watching them. Were the students aware of the performance gap when they answered this question? It seems that the group who never had the opportunity to produce a video preferred traditional classes which might simply reflect their lack of exposure to the other methodology.\nThe authors do not discuss the failure of the intervention to improve scores on the final exam. This is worth commenting on. One proposed benefit of active learning is improved long-term retention. If retention was not improved, active learning may not have been successful. The related issue with this study was that the intervention only covered 20% of the course. Any benefit on the final exam could have been diluted by the other components of the course.\nPerformance of the 2 groups on the first exam was different. Is there any explanation for this difference? The passive learning group subsequently performed worse on the second test than on the first test (while the active group improved). Can the authors comment on this? Was the post-test simply more difficult, or do they believe there was another reason for the control group to perform worse on the post-test?\nI would suggest that the authors include a Limitations section. They should note the lack of validated assessment tool (if that is the case), the inability to distinguish which part of their intervention was responsible for the improved learning (active format or technology or perhaps even absence of instructor). They should also mention their inability to comment on the effects of their intervention on long-term retention of learning.\nOn the basis of this study, what are the authors’ recommendations for educators and for education researchers?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1107
https://f1000research.com/articles/8-129/v1	30 Jan 19	{ "type": "Method Article", "title": "Enhancing gene set enrichment using networks", "authors": [ "Michael Prummer" ], "abstract": "Differential gene expression (DGE) studies often suffer from poor interpretability of their primary results, i.e., thousands of differentially expressed genes. This has led to the introduction of gene set analysis (GSA) methods that aim at identifying interpretable global effects by grouping genes into sets of common context, such as, molecular pathways, biological function or tissue localization. In practice, GSA often results in hundreds of differentially regulated gene sets. Similar to the genes they contain, gene sets are often regulated in a correlative fashion because they share many of their genes or they describe related processes. Using these kind of neighborhood information to construct networks of gene sets allows to identify highly connected sub-networks as well as poorly connected islands or singletons. We show here how topological information and other network features can be used to filter and prioritize gene sets in routine DGE studies. Community detection in combination with automatic labeling and the network representation of gene set clusters further constitute an appealing and intuitive visualization of GSA results. The RICHNET workflow described here does not require human intervention and can thus be conveniently incorporated in automated analysis pipelines.", "keywords": [ "differential gene expression analysis", "gene set analysis", "enrichment analysis", "network analyis", "GSEA" ], "content": "Introduction\n\nInterpretation of whole-transcriptome differential expression studies is often difficult because the sheer volume of the differentially expressed genes (DEGs) can be overwhelming. It is common place in designed experiments with more than just a marginal biological effect to find several thousands of differentially expressed genes (DEGs). One way to handle the vast numbers and to identify the biological consequences of gene expression changes is to associate them with overarching processes involving a whole set of genes, such as GO terms or KEGG pathways.\n\nCurated genesets have been designed or discovered for a wide range of common contexts, such as, a biological process, molecular pathway, or tissue localization1,2. They have been introduced in the past not only to reduce complexity and to improve interpretability but also to increase statistical power by reducing the number of performed tests. As it turns out, this often results in finding hundreds of differentially regulated pathways1.\n\nAs with co-expressed genes, many of the pathways exhibit strong mutual correlation because they contain a large proportion of shared genes which is in turn a result of the fact that many of them describe closely related aspects of an overarching biological theme. Therefore, to further increase interpretability of differential geneset regulation and to capture the global change of a biological phenotype, it would be desirable to identify possibly existing umbrella organizations among genesets.\n\nNetworks are ideal to model dependencies, interactions, and similarities among individuals3–5, be it people, computers, genes, or genesets. The degree of connectivity between them can have an influence on information flow and defines communities or cliques, i.e., clusters of highly connected nodes within and infrequent connections between them.\n\nIn order to construct a geneset network, a similarity measure is required and can be defined as the fraction of common genes, also called the Jaccard index6. Other ways to measure similarity among genesets include, for instance, coexpression strength as implemented in WGCNA7,8.\n\nCommunity detection based on network topology is a standard problem in the analysis of social networks9,10. Well-established algorithms allow for computationally efficient clustering of genesets and can be used to identify highly connected sub-networks. There is no unique or optimal method available but many options exist. Popular methods to define clusters include the edge-betweenness criterion, the Infomap or the Louvain algorithm (igraph), as well as hierarchical or kmeans clustering.\n\nOnce geneset clusters are defined they can be characterized by their size and connectivity and thus prioritized and ranked. In particular, the clusters can be categorized as singletons, doublets, medium and large or dense and loose clusters.\n\nNetwork analysis not only allows for detection of clusters and performance of measurements on them, networks are also straightforward and appealing visualizations of similarities among genesets. There are a couple of interactive visualization software tools available, of which Cytoscape is probably the most popular11. In some cases interactivity is useful but the emphasis here is to provide some of Cytoscape’s features without any human intervention for easy integration into automatic analysis pipelines. For instance, automatic labeling of communities using the n most frequent terms was adopted here, similar as in Kucera et al.12.\n\nThe purpose of this step-by-step workflow is to provide a fully automated and reproducible procedure for downstream analysis and visualization of differential geneset analysis results in R13. The focus is on supporting scientists in result interpretation by bringing order into the list of differentially regulated genesets based on biological rather than pure statistical arguments. The workflow is suitable for any kind of geneset library including new or custom sets and any kind of geneset analysis method.\n\nStarting with differential expression analysis of a model dataset, geneset analysis is performed based on the MSigDB library. A geneset network is constructed to identify isolated genesets (singletons) and geneset pairs (doublets). Larger connected sub-networks are then split into smaller clusters of closely related genesets describing similar processes. The effect of each modification step on the network topology is visually documented in Figure 1–Figure 4. Using the most frequently occurring terms in the geneset names of a cluster, an attempt to automatically assign cluster labels is made. Finally, all labeled clusters of genesets are plotted to provide a one page overview of the results.\n\n\nPreparations\n\nThe packages required for this workflow provide plotting functions (ggplot2 and relatives), network functions igraph14 and GGally, text analytics functions (wordcloud, etc.) and gene expression analysis functions DESeq215, limma16, and org.Hs.eg.db.\n\n\n\nIn addition to and often based on igraph, several R packages for network visualization are available and described in the form of tutorials17,18.\n\nWe are using the popular airway data set19 and perform a simple differential expression analysis.\n\n\n\nWe are using the popular org.Hs.eg.db package based on the UCSC annotation database and keep only genes with a unique mapping.\n\n\n\nWe are using the popular KEGG, Reactome, and Biocarta pathways from the MSigDB gene set library C2. The following chunk guarantees that the gene set library list object is called gset.\n\n\n\nCompetitive gene set enrichment analysis is performed using the function camera() from the limma package. We include uni-directional and bi-directional enrichment by using both the test statistics (“up” or “down”) and its modulus (“mixed”) for gene set testing. We limit the following network analysis to gene sets with a FDR < 0.05.\n\n\n\nStarting from 1077 gene sets, 264 are found to be differentially regulated. Many of them are expected to describe similar processes and to be highly correlated.\n\n\nNetwork construction\n\nWe construct a gene set network based on the proportion of common genes as the inverse distance measure. The nodes are gene sets which are connected by edges if the Jaccard index\n\n\n\nis larger than a preset threshold, J > 0.2. While this threshold is somewhat arbitrary it has proven to be a reasonable one in many projects. Nevertheless, it is strongly recommended to investigate its effect on the quality of the results.\n\n\n\nThe Jaccard matrix, or adjacency matrix, can be conveniently used to construct a network object using the function igraph::graph_from_adjacency_matrix(). In this example geneset, similarity is measured using all member genes irrespective of whether they were detected and present in the data. Alternatively, one could include only genes present in the data depending on whether the current data seem more relevant and trustworthy or the prior information given by the geneset definition. Graphical display is achieved here using ggnet::ggnet2() (Figure 1).\n\n\n\nNode colors indicate whether the member genes of a set are predominantly up or down regulated or whether there is no preferential direction (mixed).\n\n\nNetwork modifications\n\nIn the following, components of the network for which network analysis does not improve interpretability are identified and put to aside. This includes singletons, i.e., genesets not connected to any other geneset, and doublets, also termed binary systems or dumbbells, i.e., pairs of genesets connected with each other but isolated from the rest.\n\n\n\nIn total, 49 singletons were identified and excluded from further analysis (Table 1). It is important to note that these genesets, while down-prioritized for the time being, may still be worthwhile investigating later.\n\n\n\nThe color scheme is the same as above. The node size corresponds to the number of genes in a set.\n\nFigure 2 shows the remaining network clusters, with the size of the nodes representing the number of genes in the set.\n\nNext we also want to separate clusters with less than 3 gene sets. To do so, we separate disjoint subnets as individual objects, count their members, and delete all vertices belonging to clusters of size smaller than 3.\n\n\n\nIn Table 2, consecutively listed gene sets with the same id belong to the same binary cluster. Often these are gene sets from different libraries describing the same biological process or phenotype. In total, 16 binary clusters were identified, for which network analysis would not be useful.\n\n\n\nColored according to disjoint subnetworks.\n\nWithout singletons and binary clusters, we are left with larger disjoint subnets (Figure 3).\n\nThe larger disjoint clusters may consist of so-called communities, i.e., sub-networks of highly inter-connected nodes that stick together by only one or a few edges. We are using the popular edge betweenness property to identify these community-connecting edges and remove them in order to split large clusters into smaller ones.\n\n\n\nThe result of this network-based clustering is shown in Figure 4\n\n\nAutomatic annotation of gene set clusters\n\nIn analogy to the popular interactive network visualization tool cytoscape12, we attempt to generate automatic labels for gene set clusters. Gene set names are split into individual words and counted within each cluster. The four most frequent terms occurring at least twice are used as labels. The function clust_head() is defined for this purpose and contains an exclusion list of words not used.\n\n\n\nThere are many possibilities to visualize geneset clusters and often a compromise between information content and crowding has to be found. Here, we are producing a lattice of network plots, one for each sub-net, with the automatic annotation as title (Figure 5). We begin by generating the cluster titles using the clust_head() function followed by cleaning up and ordering by cluster size.\n\n\n\nThen we generate a list of ggplot objects, one for each cluster or sub-net. For smaller sub-nets, the nodes are labelled with the first 4 words of their names; the first word was removed before as it is usually the name of the geneset library. For larger sub-nets, this is not feasible without overprinting. Titles are missing if none of the words from the geneset names occurred more than once.\n\n\n\n\n\nOnly the first 16 connected subnets are shown. Geneset labels are omitted for clusters with more than 5 members.\n\n\nDiscussion\n\nWe have presented an automated workflow based on a small number of R packages for prioritization and visualization of gene set analysis results using networks, which we call RICHNET. We demonstrated how community detection facilitates categorization of differentially regulated gene sets into singletons and clusters of different size ranges. Automated label generation allowed to associate these clusters with biological themes or processes of which the member gene sets are part of.\n\nThe RICHNET workflow could be altered or extended quite naturally in a number of ways but the version presented here is the one we typically apply in our research service projects. One advantage over other approaches is that it does not depend on a particular geneset library. Specific hierarchically constructed genesets, such as GO terms, would offer a straightforward way to arrive at a more global process description using higher levels in their tree structure. A second advantage is that it does not depend on the existence of a good quality gene or protein interaction network for the particular organism or disease state which is often not feasible. Only very few genesets are network-based (e.g. KEGG pathways) and would thus offer a straight-forward way to use an a priori network topology. Thirdly, similar as in reference 8, a geneset similarity network could be constructed in the form of a co-enrichment network from GSVA enrichment scores20 using weighted co-expression network analysis (WGCNA)7. However, this approach relies on a relatively large sample size whereas the sample size requirement of RICHNET is not more than the GSA it relies on.\n\nAs an alternative to the networks of genesets described here, networks of genes could be created in a reciprocal way. The underlying similarity metric between genes could be defined as the proportion of common genesets among all genesets they are part of. This approach would be equivalent to a STRING-DB network with “databases” as the only interaction allowed21.\n\nOne possible future extension of the RICHNET workflow could be the introduction of a consensus similarity metric from multiple initial networks and different community detection or cluster algorithms to improve stability against noise. A second avenue forward could be the introduction of interactive graphics in 2D or 3D17 to allow moving, pulling, rotation or zoom and display of node specific or edge specific information.\n\nSome may argue in favor of encapsulating the RICHNET workflow in an R or Bioconductor package. However, it is our strong believe that for the sake of transparency and given the straightforward nature of the code it serves better to publish it openly. This way we encourage the users to adapt it to their specific requirements, to improve and expand on it.\n\n\nData availability\n\nThe data used in this workflow is included in the airway R-package19.\n\n\nSoftware availability\n\nThe R markdown file for this workflow is available at: https://doi.org/10.5281/zenodo.253916313.\n\nLicense: Creative Commons CC BY license.\n\nThis workflow depends on various packages from version 3.7 of the Bioconductor project, running on R version 3.5.0 or higher. A complete list of the packages used for this workflow is shown below:\n\n", "appendix": "Author contributions\n\n\n\nMP conceptualized the content, developed the method, performed the analysis and wrote the manuscript.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe author would like to thank all members of NEXUS and in particular Daniel Stekhoven for fruitful discussions as well as Beate Sick (UZH) and Phil Cheng (USZ) for critically reading the manuscript.\n\n\nFootnotes\n\n1The terms geneset and pathway are used interchangeably throughout this document and refer to a set of genes.\n\n\nReferences\n\nRouillard AD, Gundersen GW, Fernandez NF, et al.: The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database (Oxford). 2016; 2016: pii: baw100. 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\nBarabási AL, Oltvai ZN: Network biology: understanding the cell's functional organization. Nat Rev Genet. 2004; 5(2): 101–113. PubMed Abstract \| Publisher Full Text\n\nVidal M, Cusick ME, Barabási AL: Interactome networks and human disease. Cell. 2011; 144(6): 986–998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIdeker T, Krogan NJ: Differential network biology. Mol Syst Biol. 2012; 8(1): 565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerico D, Isserlin R, Stueker O, et al.: Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One. 2010; 5(11): e13984. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLangfelder P, Horvath S: WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9(1): 559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorsson V, Gibbs DL, Brown SD, et al.: The Immune Landscape of Cancer. Immunity. 2018; 48(4): 812–830.e14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGirvan M, Newman ME: Community structure in social and biological networks. Proc Natl Acad Sci U S A. 2002; 99(12): 7821–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBedi P, Sharma C: Community detection in social networks. Wiley Interdisciplinary Reviews: Data Mining and Knowledge Discovery. 2016; 6(3): 115–135. Publisher 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): 2498–504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKucera M, Isserlin R, Arkhangorodsky A, et al.: AutoAnnotate: A Cytoscape app for summarizing networks with semantic annotations [version 1; referees: 2 approved]. F1000Res. 2016; 5: 1717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichael P: Enhancing gene set enrichment using networks. zenodo. 2019. http://www.doi.org/10.5281/zenodo.2539163\n\nCsardi G, Nepusz T: The igraph software package for complex network research. InterJournal. Complex Sy:1695, 2006. Reference Source\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRitchie ME, 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\nOgnyanova K: Static and dynamic network visualization with R. 2015. Reference Source\n\nTyner S, Briatte F, Hofmann H: Network Visualization with ggplot2. R Foundation for Statistical Computing. 2017; 9(1): 27–59. Reference Source\n\nHimes BE, Jiang X, Wagner P, et al.: RNA-Seq transcriptome profiling identifies CRISPLD2 as a glucocorticoid responsive gene that modulates cytokine function in airway smooth muscle cells. PLoS One. 2014; 9(6): e99625. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHänzelmann S, Castelo R, Guinney J: GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics. 2013; 14(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzklarczyk D, Morris JH, Cook H, et al.: The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res. 2017; 45(D1): D362–D368. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "44249", "date": "07 Mar 2019", "name": "Kimberly Glass", "expertise": [ "Reviewer Expertise computational biology", "systems biology", "network biology", "network 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\nIn \"Enhancing gene set enrichment using networks\" the author describes a pipeline to visualize the gene sets associated with a particular differential-expression analysis as a network. In this network connections between gene sets are based on common/shared gene annotations. The paper is clearly written and the decisions made in the pipeline are reasonable. There are only a few points on which I would like to see more discussion:\nIn the introduction the authors mention the Jaccard index as a similarity measure (alongside coexpression of genes in WGCNA). There are many similarity measures: hamming distance, cosine similarity, Fisher's exact test, as well as many other measures for continuous variables that can easily be adapted to binary variables. Which one of these is used to construct the gene-set network could impact the structure of the network. The pros and cons of using the Jaccard index as well as other similarity measures warrants more discussion (e.g. some may better capture relationships between terms with many gene annotations, and some may better capture relationships between terms with fewer gene annotations).\n\nGO terms are structured as a DAG, with genes annotated to child terms propagating to parent terms. This underlying structure will impact the structure of the similarity network between GO terms, and is worth pointing out in the manuscript.\n\nIn the introduction, the authors state \"the clusters can be categorized as....medium and large or dense and loose clusters\". The author should either include more discussion about how these can be quantified (i.e. what is a \"medium\" cluster) or they should illustrate the quantification of these categories in their example.\n\nThe equation “J=NumberofcommongenesNumberofallgenes” looks mis-formatted. It also would be better presented as: “J = intersect(set A, set B)/union(set A, set B)”.\n\nThe authors should also consider the structure of a general gene-set network (one not restricted to gene-sets associated with differentially-expressed genes). It is possible that the singletons/doublets that the authors remove may simply come from a sparser area of this \"general\" gene-set network (and the clusters a denser area), in which case the pruning step is removing relevant results (while retaining less informative ones that might be picked up by chance).\n\nIn naming the clusters, I would suggest normalizing the number of instances of a word against its frequency in the entire database. For example \"cell\" is a much more common word in KEGG/GO term names than \"glycolosis\". From a biological point of view, if all the terms that contain \"glycolosis\" are in the same cluster (even if it’s only 1-2 terms), this is likely much more interesting to highlight than if \"cell\" appears frequently in that cluster (but also in many other terms outside of the cluster).\n\nDo the authors have any thoughts about how to interpret clusters with \"missing titles\" (no word appearing more than once)?\nMinor comments:\nBe sure to spell out the GO and KEGG acronym for first usage. Some of the longer names in Figure 5 appear truncated (e.g. \"cell death signalling via\").\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": "4735", "date": "16 Jul 2019", "name": "Michael Prummer", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your constructive comments. Below are point-by-point responses to the individual comments. While a discussion on the influence of different distance metrics on network structure is interesting it is not in the scope of this work. Sidenote: a discussion on the influence of different community detection algorithms would be interesting as well. These aspects are critical to any attempt at clustering data but it is assumed here that the qualified reader and user of this workflow is aware of it. The issue is briefly mentioned in paragraph four of the discussion. Avoiding the use of GO terms was an attempt to avoid having to open up the discussion about the influence of their hierarchical structure on results. I am convinced this would make the discussion unnecessarily complicated and that it is better done elsewhere. The equation defining the Jaccard index is formatted correctly in the PDF. In the manuscript it is emphasized that nothing is removed or deemed irrelevant and that putting aside singletons and doublets is just a means of sorting. Indeed, an unusually large proportion of singletons may indicate an unexplored area of biology. In such a situation, relying on common knowledge in the form of published genesets may not be the wisest way to go at all. There are a number of different possibilities to obtain a representative label for a cluster and in this manuscript a relatively simple one was chosen. It may not be the most sophisticated but it is straight forward to understand. The following text is included in section Lattice of annotated networks on missing titles: “This may be indicative for a semantically mixed cluster or for sparse prior knowledge.”" } ] }, { "id": "44250", "date": "13 Mar 2019", "name": "Monther Alhamdoosh", "expertise": [ "Reviewer Expertise Bioinformatics and AI. Developed gene set analysis method", "which is widely used by the research community." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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\nThis workflow paper proposes an interesting approach for prioritizing gene sets in gene set analysis by utilizing network-based algorithms. The author presented a supposedly working code that is reproducible. They proposed focusing on communities of gene sets in order to identify gene sets that are more relevant to the conditions under study. Nonetheless, they show how to explore singletons and binary systems in the list of significant gene sets. Moreover, natural language processing methods were used to annotate the clusters of gene sets. I found this particular extension very useful to summarize gene sets analysis results. However, I didn't get that far in running the code. Please see my comments below for improvements.\n\nMajor comments\nAdd code to install pre-requisite packages. In my case I had to run the following command to obtain missing packages:\ninstall.packages(c(\"cowplot\", \"ggrepel\", \"kableExtra\", \"igraph\", \"GGally\", \"wordcloud\", \"tm\", \"SnowballC\"))\nAdd code to install the airway experiment package BiocManager::install(\"airway\", version = \"3.8\") I managed to reproduce the analysis up to the point of generating the first network. First, it was required to install the c(\"network\", \"sna\", \"scales\") packages, which was not explained in the text. Then, error raised while invoking ggnet2:\n\nError in ggnet2(net, size = 2, color = \"Direction\", palette = palette, : could not coerce net to a network object\n\nNot sure whether related to the version of R. This is my R session:\n\nR version 3.5.0 (2018-04-23) Platform: x86_64-pc-linux-gnu (64-bit) Running under: RHEL\nMatrix products: default BLAS:**ps/r/3.5.0/lib64/R/lib/libRblas.so LAPACK: **/apps/r/3.5.0/lib64/R/lib/libRlapack.so\nlocale: [1] LC_CTYPE=en_US.UTF-8\n\nLC_NUMERIC=C\n\nLC_TIME=en_US.UTF-8\n\n[4] LC_COLLATE=en_US.UTF-8\n\nLC_MONETARY=en_US.UTF-8\n\nLC_MESSAGES=en_US.UTF-8\n\n[7] LC_PAPER=en_US.UTF-8\n\nLC_NAME=C\n\nLC_ADDRESS=C\n\n[10] LC_TELEPHONE=C\n\nLC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C\n\nattached base packages: [1] parallel stats4\n\nstats\n\ngraphics grDevices utils\n\ndatasets methods\n\nbase\n\nother attached packages: [1] airway_1.2.0\n\nSnowballC_0.6.0\n\ntm_0.7-6\n\n[4] NLP_0.2-0\n\nwordcloud_2.6\n\norg.Hs.eg.db_3.7.0\n\n[7] AnnotationDbi_1.44.0\n\nlimma_3.38.2\n\nDESeq2_1.22.1\n\n[10] SummarizedExperiment_1.12.0 DelayedArray_0.8.0\n\nBiocParallel_1.16.0\n\n[13] matrixStats_0.54.0\n\nBiobase_2.42.0\n\nGenomicRanges_1.34.0\n\n[16] GenomeInfoDb_1.18.1\n\nIRanges_2.16.0\n\nS4Vectors_0.20.1\n\n[19] BiocGenerics_0.28.0\n\nGGally_1.4.0\n\nigraph_1.2.4\n\n[22] kableExtra_1.0.1\n\nknitr_1.20\n\nreshape2_1.4.3\n\n[25] ggrepel_0.8.0\n\ncowplot_0.9.4\n\ngplots_3.0.3\n\n[28] ggplot2_3.1.0\n\nRColorBrewer_1.1-2\n\nloaded via a namespace (and not attached): [1] bitops_1.0-6\n\nbit64_0.9-8\n\nwebshot_0.5.1\n\nhttr_1.3.1\n\n[5] rprojroot_1.3-2\n\ntools_3.5.0\n\nbackports_1.1.2\n\nR6_2.3.0\n\n[9] rpart_4.1-13\n\nKernSmooth_2.23-15\n\nHmisc_4.1-1\n\nDBI_1.0.0\n\n[13] lazyeval_0.2.1\n\ncolorspace_1.4-0\n\nnnet_7.3-12\n\nwithr_2.1.2\n\n[17] tidyselect_0.2.5\n\ngridExtra_2.3\n\nbit_1.1-14\n\ncompiler_3.5.0\n\n[21] rvest_0.3.2\n\nhtmlTable_1.12\n\nnetwork_1.14-377\n\nxml2_1.2.0\n\n[25] slam_0.1-45\n\ncaTools_1.17.1.1\n\nscales_1.0.0\n\ncheckmate_1.8.5\n\n[29] readr_1.1.1\n\ngenefilter_1.64.0\n\nstringr_1.3.1\n\ndigest_0.6.18\n\n[33] foreign_0.8-71\n\nrmarkdown_1.10\n\nXVector_0.22.0\n\nbase64enc_0.1-4\n\n[37] pkgconfig_2.0.2\n\nhtmltools_0.3.6\n\nhtmlwidgets_1.3\n\nrlang_0.3.0.1\n\n[41] rstudioapi_0.8\n\nRSQLite_2.1.1\n\nbindr_0.1.1\n\nstatnet.common_4.2.0\n\n[45] gtools_3.8.1\n\nacepack_1.4.1\n\ndplyr_0.7.8\n\nRCurl_1.96-0\n\n[49] magrittr_1.5\n\nGenomeInfoDbData_1.2.0 Formula_1.2-3\n\nMatrix_1.2-15\n\n[53] Rcpp_1.0.0\n\nmunsell_0.5.0\n\nstringi_1.2.4\n\nyaml_2.2.0\n\n[57] zlibbioc_1.28.0\n\nplyr_1.8.4\n\ngrid_3.5.0\n\nblob_1.1.1\n\n[61] gdata_2.18.0\n\ncrayon_1.3.4\n\nlattice_0.20-38\n\nsplines_3.5.0\n\n[65] annotate_1.60.0\n\nhms_0.4.2\n\nsna_2.4\n\nlocfit_1.5-9.1\n\n[69] pillar_1.3.0\n\ngeneplotter_1.60.0\n\nXML_3.99-0\n\nglue_1.3.0\n\n[73] evaluate_0.12\n\nlatticeExtra_0.6-28\n\ndata.table_1.11.8\n\ngtable_0.2.0\n\n[77] purrr_0.2.5\n\nreshape_0.8.8\n\nassertthat_0.2.0\n\nxtable_1.8-3\n\n[81] coda_0.19-2\n\nviridisLite_0.3.0\n\nsurvival_2.43-1\n\ntibble_1.4.2\n\n[85] memoise_1.1.0\n\nbindrcpp_0.2.2\n\ncluster_2.0.7-1\n\nThere are 58 gene sets that are significant in both the uni-directional and bi-directional tests. The former was assumed to be the determinant of the direction of the gene set, which is a bit confusing here. It would be good if the author could elaborate on the reasoning behind assigning the directional status. It would be good to highlight the relationship between the identified clusters and annotations, and the underlying experimental conditions.\n\nMinor comments\nEGSEAdata experiment package can be used to load huge number of gene sets including MSigDB gene sets. This is an example:\n\nlibrary(EGSEAdata) library(EGSEA) gset = buildMSigDBIdx(entrezIDs = res$entrezgene, species = \"human\", geneSets = \"c2\", min.size = 3) idx = gset$c2@idx gs.libs = sapply(names(idx), function(x) strsplit(x, \"_\")[[1]][1]) idx = idx[which(gs.libs %in% c(\"KEGG\", \"REACTOME\", \"BIOCARTA\"))]\nPlease see this workflow paper for more information1.\n\nThe workflow is tailored towards the camera analysis. What if users want to use this workflow for other GSA methods? A more generic example is needed. Any possibility to wrap the network analysis code and make it easier for users to invoke as a function? Probably, an R package named RICHNET can be developed along with this wonderful workflow.\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "4734", "date": "16 Jul 2019", "name": "Michael Prummer", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your constructive comments. Below are point-by-point responses to the individual comments. The missing packages were included in the initial package installation and loading part. The source of the error in ggnet2 is fixed. Uni- and bi-directional tests are preformed in parallel. Afterwards, priority is given to the uni-directional test as it is more stringent (either up- or down-regulated genes). The bi-directional case is biologically meaningful as well as an upregulation of and inhibitory member of a pathway has the same effect as the downregulation of an activatory member. But all these details are related to one particular choice of enrichment analysis whereas the extent of this work starts after a list of candidate genesets was generated by any appropriate method. Biological interpretation of the geneset clustering results produced here is beyond the scope of this work. A code chunk using EGSEAdata to build the geneset library was included in the manuscript. The following sentence is included in paragraph 2 of the discussion: “One advantage over other approaches is that it does not depend on a particular geneset library or geneset analysis method. Any means of selecting genesets of interest can be used.”" } ] }, { "id": "45391", "date": "02 Apr 2019", "name": "Jill P. Mesirov", "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\nWhile the author does address an important general issue of downstream analysis of gene set enrichment results, his approach is almost identical to “Enrichment Map: A Network-Based Method for Gene-Set Enrichment Visualization and Interpretation” (Merico et al., 20101). Furthermore, Enrichment Map provides much more information in its visualization including the size of the gene set (via the size of the node); the level of enrichment of the gene sets (via a color gradient from blue for down- to red for up-regulation); as well as the extent of overlap between gene sets (via the thickness of the edge connecting gene sets using the Jaccard index as a weight). Even considering the manuscript independent of this issue, there are a number of other concerns with the study (some minor but some more major) that require attention from the author and a major revision of the manuscript. They are listed more or less in the order that the issue appears in the manuscript.\nThe author claims that gene set analysis “often results in finding hundreds of differentially regulated pathways”. What is the basis for this claim? While it is true that this may occur – it is also true that there may also be no statistically significantly differentially regulated pathways. Has he done experiments on a large number of benchmark data sets to establish this claim of “often”? Moreover, this may be a result of the particular gene set collection used for the analysis. Is it a function of the particular data set or enrichment approach he is using (CAMERA)? We note here that the author never describes the measure CAMERA uses for enrichment nor how up/down is determined. All these open issues should be addressed.\n\nThe use of “umbrella” organization is somewhat unclear – do you mean hierarchy? Note also the need for a hierarchy of the significantly activated or repressed pathways may result from the author’s choice of gene set collection, which may not provide a significant or specific enough result. This point should be addressed.\n\nThe author provides little explanation for the arbitrary choice of Jaccard Index > 0.2 as the connectivity threshold. Additionally, as the edges in the networks produced by this method do not represent any other kind of continuous data, has the author considered weighting the edges by the Jaccard index in the visualization to allow the user to see the effect of various thresholds? In fact, Enrichment Map (cited above) uses the Jaccard Index itself to determine the weight of the line connecting two overlapping gene sets. These approaches need to be compared and the choice of threshold better explained and justified.\n\nPrioritizing and ranking the active pathways/gene sets by number of gene sets in a network hub and degree of connectivity seems inadequate if one is looking for biological insights. A biological measure of prioritization would be preferable which includes the levels of activation. Often a study is not done in a vacuum and so in fact some signals may be expected – this may serve as an additional measure for prioritization and even validation of the method. This issue is not addressed and should be and some validation of the results of the only test set analysed should be supplied.\n\nIt is important to include and describe the numerous network-based enrichment methods that have been published and specifically Enrichment Map (cited above) which is a plugin to both Cytoscape and GSEA and does exactly what the author is describing (downstream analysis of gene set enrichment results) but is never mentioned or referenced. At the very least its performance should be compared to the author’s method. This is a requisite for any newly proposed method.\n\nThe goal of this method is to “focus is on supporting scientists in result interpretation by bringing order into the list of differentially regulated gene sets based on biological rather than pure statistical arguments.” But one might ask why do this based on network measures of association and the visualization is very sparse in what it represents as noted above. Also, if this is the goal – it is incumbent on the author to show why this works better than other existing methods (see above) or even using a more sensitive/specific collection of gene sets, e.g., the Hallmark collection in MSigDB (Liberzon et al., 20152). This provides a collection of sets for which essentially this work has been done with additional biological curation. Comparison with other methods and with the use of other gene set collections should be included.\n\nThe author’s example on the “airway” data set employs an analysis with KEGG, Reactome, and BioCarta. Using these 3 databases together means there will be a large amount of redundancy and overlap in the gene sets he uses – again it would be important to compare his results using these collections against the Hallmark collection. Furthermore – why just the results from one data set? The method would be better tested against multiple data sets – some where the signal is very strong and some where the signal is weaker.\n\nThe author uses the CAMERA method for testing gene sets. This method produces only a binary “Up” or “Down” measurement of enrichment which is used to color the nodes in the resulting network visualizations. This is a very coarse way of testing gene sets. The interpretability of the network visualizations could be improved by using a method such as GSEA, which gives a continuous enrichment score, and coloring the nodes with a gradient to compare degrees of up- or down-regulation and weighting the edges as is done in Enrichment map as noted above.\n\nFinally – after application of RICHNET the author only describes and discusses the nature of his resulting networks. There is no discussion that we could see of the biological insights gained, how realistic they were, whether they recapitulated known signals in the data set, etc.\n\nTechnical concerns with the code as presented: The analysis fails with an unintuitive error (“could not coerce net to a network object”) if the library “Intergraph” is not installed. While this library is listed in the sessionInfo() printout in the manuscript, this library should be included in the first library() cell of the notebook since its absence is not immediately obvious given this error.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\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? No", "responses": [ { "c_id": "4732", "date": "16 Jul 2019", "name": "Michael Prummer", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your critical comments. Below are point-by-point responses to the individual comments.The remark on the similarity of this work with prior work by others is fully justified and is discussed in the manuscript. The mentioned work by Merico et al. is included as reference 6.The open nature of this workflow script allows for a straightforward implementation of any additional graphical feature should the user of this workflow wish to do so.By using the term \"often\" I was hoping to emphasize that while the statement is true for more than a small number of cases there may be cases where the statement is wrong. How often the statement is true or whether it depends on the choice of geneset or enrichment algorithm or FDR cutoff is less important as long as there exist enough cases for which the workflow presented here may be useful.Most geneset collections consist of largely overlapping sets which describe very similar processes or properties. The workflow presented here was made for such cases. For the workflow to be useful, an explicit hierarchy, such as with GO-terms, is not required.The network as constructed here has indeed edges weighted by the Jaccard index. Visualization by varying edge thickness has proven to be difficult to discriminate in my hands. The open nature of this workflow script should allow for an easy implementation of this feature should a user wish to do so.To address the choice of the Jaccard index cutoff the following sentence has been added at the end of section Network construction: “As a guide for finding a reasonable threshold a broad distribution of disjoint cluster sizes is desired. Network analysis does not help if the cutoff is too large (no connections) or too small (all sets are connected with each other).”One measure of quality of the network construction and community detection is the semantic purity of the clusters. This can be easily seen for the binary systems (and is discussed there) and to a lesser extent for the larger clusters. Whether biological insight can be gained depends strongly on the choice of the genesets in relation to the field of study. RICHNET provides a means of grouping and organizing results and does not generate them.Enrichment Map is indeed very similar and a perfectly fine interactive tool (see reference 6 of the manuscript). The present workflow implements a similar functionality in R and allows for integration in automated analysis pipelines.Many of our clients find the hallmark geneset too generic and prefer more diverse geneset collections, such as, KEGG or Reactome.Your suggestion to include more test results with respect to different geneset libraries, Jacquard score cutoffs, community detection algorithms, and datasets is well received. We are planning to do this in the future.Using the negative log of the enrichment p-value as enrichment score has been demonstrated in numerous cases in the literature and replacing the categorical colour scale by a continuous one is possible.Biological interpretation of the geneset clusters produced here is beyond the scope of this work.The package intergraph was included in the library installation and loading part." } ] }, { "id": "45393", "date": "10 Apr 2019", "name": "Rita Casadio", "expertise": [ "Reviewer Expertise Functional annotation of protein variants", "machine learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an efficient and very useful method for gene set analysis. Their approach is network based and facilitates retrieval of sets of genes that are highly connected. Grouping is due to common context (molecular pathways, biological function, tissue localisation). Furthermore, this procedure facilitates gene enrichment and the identification of singletons or poorly connected islands.\nThe RICHNET workflow stands as a user friendly workflow that can be easily incorporated in automated analysis pipelines.\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": "4739", "date": "16 Jul 2019", "name": "Michael Prummer", "role": "Author Response", "response": "Thank you for reviewing the manuscript and for your very positive comments." } ] } ]	1	https://f1000research.com/articles/8-129
https://f1000research.com/articles/8-183/v1	14 Feb 19	{ "type": "Review", "title": "How ‘smart’ is smart dentistry?", "authors": [ "Peter Kokol", "Helena Blažun Vošner", "Jernej Završnik", "Marko Turčin", "Helena Blažun Vošner", "Jernej Završnik", "Marko Turčin" ], "abstract": "Background: Latest advances in information and health technologies enabled dentistry to follow the paradigm shift occurring in medicine – the transition to so called smart medicine. Consequently, the aim of this paper is to assess how ‘smart’ is smart dentistry as of the end of 2018. Methods: We analysed the state of the art in smart dentistry, performing bibliometric mapping on a corpus of smart dentistry papers found in the Scopus bibliographical database. Results: The search resulted in a corpus of 3451 papers, revealing that smart dentistry research is following the progress in smart medicine; however, there are some gaps in some specific areas like gamification and use of holistic smart dentistry systems. Conclusions: Smart dentistry is smart; however, it must become smarter.", "keywords": [ "smart medicine", "smart dentistry", "bibliometric mapping", "papers as subjects" ], "content": "Introduction\n\nAdvances in information, communication and health technologies triggered a paradigm shift in modern medicine – the transition to so called smart medicine. Some of the first appearances of the term smart medicine in the above context appeared in the late eighties and ninetieths in relation to (1) smart medical systems in the Space Station1, (2) nuclear medicine and surgery2 and (3) advanced biomimetic materials3. In the beginning of the third millennia the research literature production on this subject started to grow. New smart application were introduced, like robotics surgery4, smart medical systems in nutrition5, smart medical records6 and smart sensors7. Recently, additional new smart health technologies including personalized and precision medicine, gamification based treatment, artificial intelligence, 3D printing, nanotechnology, Internet of Things and semantic health records have emerged8–10. The frequency of use of the above technologies in dentistry has been used to assess the dentistry “smartness” in our study. Recently, dentistry started to follow smart medicine trends11 and the aim of this paper is to assess the ‘smartness’ of smart density using a bibliometric approach.\n\n\nMethods\n\nTo analyse the state of the art in smart dentistry, we analysed the corpus of papers retrieved from the Scopus bibliographical database (Elsevier, Netherlands). The search string was composed from representative keywords found in smart medicine research in the following manner: smart or personalized or precision or G4H or “artificial intelligence” or “3D print” or nanotechnology or robotic or IoT or “semantic health record”. The search was restricted to the subject area of dentistry (which in Scopus includes dentistry, endodontics, oral health, oral biology, orthodontics, prosthodontics and periodontology). We limited the search to the period beginning in 2001, when the growth of literature production on smart medicine began, up to 2018 (inclusive) and articles published in journals only. Using descriptive bibliometrics we identified the research literature production trends, most productive countries and most prolific journals.\n\nTo analyse and visualize the context of the smart density research literature we used a bibliometric mapping approach and a popular mapping tool called VOSViewer Version 1.6.9 (Leiden University, Netherlands)12,13. The outputs from VOSViewer are various types of bibliometric maps, frequently called science landscapes. Landscapes can reveal different patterns and aspects of research literature like associated or related terms/keywords, timelines, citation, country or networks and similar. In our study, the author cluster keyword landscape was induced using “Create a map based on bibliographic data” option in the opening VOSViewer menu. After selecting Scopus as the bibliographic database used and defining the names of files to be analysed we selected “Co-occurrence Author Keywords” as the type of analysis and “Full counting” as the counting method. Then we set the “Minimum number of occurrences of a keyword” to 8 occurrences. For all other parameters the default values were used. The proximity of terms indicates keyword similarity and the coloured clusters represent strongly associated keywords. Using a customized VOSViewer thesaurus file, we excluded common and statistical keywords like systematic review or meta-analysis from the analysis. We also mapped synonyms into one entity (for example cone beam computer tomography, cone-beam computer tomography, cone beam computed tomography, cone-beam computed tomography and cbct into cone-beam computer tomography). The thesaurus file is consisting of two columns, first includes the synonym and second the keyword in which the synonym should be mapped. To omit a keyword from the analysis, the second column entry is left empty.\n\n\nResults\n\nThe search was performed on 12th of December 2018 and resulted in a corpus of 2470 papers. The research literature production exhibits the linear growing trend from 2001 till 2016, namely from 46 to 198 articles per year, with the average increase of nine papers per year. In last two years the growth was still linear, however with an average increase of 78 articles per year. The productivity reached its peak in 2018 with 353 articles.\n\nThe most productive countries were United Stated of America (USA) (n=627), Germany (n=298), Brazil (n=223), Italy (n=174), United Kingdom (UK) (n=168), India (n=1266), Japan (n=120), South Korea (n=111). Switzerland (n=110) and China (n=119). The top 10 productive countries are belonging either to the G8 group or are countries with highly developed economies and health systems. The most prolific journals are Journal of Prosthetic Dentistry (n=131), Dental Materials (n=83), Oral Oncology (n=79), Journal of Oral and Maxillofacial Surgery (n=75), Journal of Dental Research (n=71), American Journal of Orthodontics and Dentofacial Orthopaedics (n=59), Clinical Oral Implants Research (n=51) and Clinical Oral Investigation (n=49). Top journals belong to the most prestigious and highest-ranking journals in the dentistry field.\n\nNine clusters (Figure 1) emerged on the cluster landscape. We used the cluster keywords as codes in the thematic analysis14, focusing on “medical smartness”. In that manner the following smart dentistry themes were derived:\n\nDigital impression (brown colour): Digital impressions represent cutting-edge technology that allows dentists to create an accurate virtual, computer-generated replica of the hard and soft tissues in the mouth using advance 3D scanning devices in a very short time. In that manner, the use of traditional impression materials that some patients find inconvenient, can be avoided. Digital optical impressions significantly increase efficiency, productivity and accuracy, and enable dentists to distribute impressions using e-mails. Digital impressions in combination with 3D print can be used to make immediate restorations, reducing the need for patients multiple office visits15.\n\nDigital dentistry in prosthodontics (yellow colour). As the name applies Digital density is focused on use of digital technologies in dentistry in general, but focusing on prosthodontics16,17, however, it also deals with smart management of patients18.\n\nDental implants and computer aided design (violet colour): The advance in dental materials required a new of design in dental practice. In that manner, computer aided design (CAD) has been introduced into dentistry19. CAD is also used for the reconstruction of face defects due to flaps or bone defects20.\n\nRobotic surgery (orange colour) is mainly used in transoral neck and head surgery21. Especially interesting is the application of robotics removal of very rare parapharyngeal space tumours22. On the other hand, computer assisted surgery is mostly used in mandibular reconstruction23.\n\nBiomaterials and nanotechnology in tissue engineering and endodontics (blue and pink colours): The idea of biomaterials in dentistry is to have a dynamic’ smart behaviour in the manner that the materials can react to changes in the environment with the advantageous changes in their properties to benefit the dental patient. These smart materials can react to stress, temperature, moisture, pH, etc. A promising version of them are bio-smart materials24. Smart materials include nanomaterials which are mainly used to fight caries, to enhance remineralization of apatite-depleted dentin, dental tissue regeneration and drug delivery25,26. On the other hand, smart brackets tend to be more efficient in reducing treatment times compared to conventional bracket, however, the quality of orthodontic treatment is similar to conventional systems as is the patient perception. An interesting recent upgrade in smart brackets is the integration of sensors, which can measure forces and moments used to improve treatment27.\n\nArtificial intelligence and precision/personal medicine in dentistry (red colour): Recently, the artificial intelligence has been introduced in dentistry to achieve the goals of precision and personalised health care28. It is used in decision making29, evaluation of facial attractiveness with maloocclusion30, diagnosing31 and similar32.\n\n3D printing in surgery, implantation and reconstruction (green and light blue colour): 3D printing has many applications in dentistry and showed improvements in precision and reduction, surgery times and personalisation33. In combination with cone beam commuted tomography34 and CAD, 3D printing has been successfully used in various endodontic challenges35.\n\nFrom a quantitative point of view, the most prolific smart medicine technologies used in dentistry are 3D printing occurring in 99 articles, nanotechnology occurring in 80 articles, robotic surgery occurring in 43 articles, digital impression occurring in 33 articles and artificial intelligence occurring in 13 articles.\n\n\nDiscussion and conclusion\n\nThe above analysis showed that smart dentistry in general is following smart medicine “movement” especially in using 3D printing, nanotechnology and smart materials, robotics, IoT (i.e. sensors) technologies, personalised and precision medicine and artificial intelligence. However, there are substantial gaps in use of smart medicine technologies regarding gamification, smart dentistry systems and semantic eHealth records. Thus, we can state the smart dentistry is smart, but it must become smarter.\n\n\nData availability\n\nOSF: Dataset 1. Smart dentistry. https://doi.org/10.17605/OSF.IO/UJRKT36\n\nLicence: CC0 1.0 Universal", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGardner RM, Ostler DV, Nelson BD, et al.: The role of smart medical systems in the Space Station. Int J Clin Monit Comput. 1989; 6(2): 91–98. PubMed Abstract \| Publisher Full Text\n\nWagner HN Jr: Nuclear medicine: the road to smart medicine and surgery. J Nucl Med. 1998; 39(8): 13N–17N, 19N, 23N-24N passim. PubMed Abstract\n\nOtero TF, Cantero I, Villanueva S: EAP as multifunctional and biomimetic materials. Proc SPIE - Int Soc Opt Eng. 1999; 3669: 26–34. Publisher Full Text\n\nDario P, Menciassi A: Robotics for surgery. 2002; 3: 1813–1814.\n\nSoller BR, Cabrera M, Smith SM, et al.: Smart medical systems with application to nutrition and fitness in space. Nutrition. 2002; 18(10): 930–936. PubMed Abstract \| Publisher Full Text\n\nSpyropoulos B, Sochos P, Tsirogiannis A, et al.: A “Smart Medical Record” for the general practitioner supporting decision making and training in cardiology. 2002; 135–136. Publisher Full Text\n\nProceedings of SPIE: Smart medical and biomedical sensor technology. 2004; 5261. Reference Source\n\nJefferson RS: Just How Smart Is Smart Medicine? MIT Scientists Are About To Find Out. Forbes. 2018; Accessed December 12, 2018. Reference Source\n\nPAH25: Smart Health Technology. Patient@Home; 2018. Accessed December 12, 2018. Reference Source\n\nŽeleznik D, Kokol P, Blažun Vošner H: Adapting nurse competence to future patient needs using Checkland’s Soft Systems Methodology. Nurse Educ Today. 2017; 48: 106–110. PubMed Abstract \| Publisher Full Text\n\nDowson T: 7 Dental Industry Trends in 2017 & What They Mean For Practice Growth. 2017; Accessed May 13, 2017. Reference Source\n\nKokol P, Završnik J, Blažun Vošner H: Bibliographic-Based Identification of Hot Future Research Topics: An Opportunity for Hospital Librarianship. J Hosp Librariansh. 2018; 18(4): 315–322. Publisher Full Text\n\nvan Eck NJ, Waltman L: Visualizing Bibliometric Networks. In: Ding Y, Rousseau R, Wolfram D, eds. Measuring Scholarly Impact: Methods and Practice. Cham: Springer International Publishing; 2014; 285–320. Publisher Full Text\n\nVaismoradi M, Turunen H, Bondas T: Content analysis and thematic analysis: Implications for conducting a qualitative descriptive study. Nurs Health Sci. 2013; 15(3): 398–405. PubMed Abstract \| Publisher Full Text\n\nFlügge TV, Schlager S, Nelson K, et al.: Precision of intraoral digital dental impressions with iTero and extraoral digitization with the iTero and a model scanner. Am J Orthod Dentofacial Orthop. 2013; 144(3): 471–478. PubMed Abstract \| Publisher Full Text\n\nMangano Guest Editor F: Digital Dentistry: The Revolution has Begun. Open Dent J. 2018; 12(Suppl-1, M1): 59–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLynch C: Defining digital dentistry: A survey of recent literature. J Dent. 2017; 59: 1. PubMed Abstract \| Publisher Full Text\n\nPirmohamed S, Bomfim DI: Utilising Digital Dentistry for the Management of Patients With Hypodontia of Lateral Incisers. Prim Dent J. 2018; 7(2): 50–55. PubMed Abstract\n\nMiyazaki T, Hotta Y: CAD/CAM systems available for the fabrication of crown and bridge restorations. Aust Dent J. 2011; 56(SUPPL. 1): 97–106. PubMed Abstract \| Publisher Full Text\n\nZweifel D, Bredell MG, Essig H, et al.: Total virtual workflow in CAD-CAM bony reconstruction with a single step free fibular graft and immediate dental implants. Br J Oral Maxillofac Surg. 2018; 56(9): 859–863. PubMed Abstract \| Publisher Full Text\n\nPoon H, Li C, Gao W, et al.: Evolution of robotic systems for transoral head and neck surgery. Oral Oncol. 2018; 87: 82–88. PubMed Abstract \| Publisher Full Text\n\nMaglione MG, Guida A, Pavone E, et al.: Transoral robotic surgery of parapharyngeal space tumours: a series of four cases. Int J Oral Maxillofac Surg. 2018; 47(8): 971–975. PubMed Abstract \| Publisher Full Text\n\nvan Baar GJC, Forouzanfar T, Liberton NPTJ, et al.: Accuracy of computer-assisted surgery in mandibular reconstruction: A systematic review. Oral Oncol. 2018; 84: 52–60. PubMed Abstract \| Publisher Full Text\n\nBadami V, Ahuja B: Biosmart Materials: Breaking New Ground in Dentistry. ScientificWorldJournal. 2014; 2014: 986912. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlenazy MS, Mosadomi HA, Al-Nazhan S, et al.: Clinical considerations of nanobiomaterials in endodontics: A systematic review. Saudi Endod J. 2018; 8(3): 163–169. Reference Source\n\nOgawa T, Saruwatari L, Takeuchi K, et al.: Ti nano-nodular structuring for bone integration and regeneration. J Dent Res. 2008; 87(8): 751–756. PubMed Abstract \| Publisher Full Text\n\nKuhl M, Gieschke P, Rossbach D, et al.: A wireless stress mapping system for orthodontic brackets using CMOS integrated sensors. IEEE J Solid-State Circuits. 2013; 48(9): 2191–2202. Publisher Full Text\n\nKearney V, Chan JW, Valdes G, et al.: The application of artificial intelligence in the IMRT planning process for head and neck cancer. Oral Oncol. 2018; 87: 111–116. PubMed Abstract \| Publisher Full Text\n\nKhanna S: Artificial intelligence: Contemporary applications and future compass. Int Dent J. 2010; 60(4): 269–272. PubMed Abstract\n\nYu X, Liu B, Pei Y, et al.: Evaluation of facial attractiveness for patients with malocclusion: a machine-learning technique employing Procrustes. Angle Orthod. 2014; 84(3): 410–416. PubMed Abstract \| Publisher Full Text\n\nJung SK, Kim TW: New approach for the diagnosis of extractions with neural network machine learning. Am J Orthod Dentofacial Orthop. 2016; 149(1): 127–33. PubMed Abstract \| Publisher Full Text\n\nMajumdar B, Sarode SC, Sarode GS, et al.: Technology: Artificial intelligence. Br Dent J. 2018; 224(12): 916. PubMed Abstract \| Publisher Full Text\n\nLouvrier A, Marty P, Barrabé A, et al.: How useful is 3D printing in maxillofacial surgery? J Stomatol Oral Maxillofac Surg. 2017; 118(4): 206–212. PubMed Abstract \| Publisher Full Text\n\nFortin T, Champleboux G, Bianchi S, et al.: Precision of transfer of preoperative planning for oral implants based on cone-beam CT-scan images through a robotic drilling machine. Clin Oral Implants Res. 2002; 13(6): 651–6. PubMed Abstract \| Publisher Full Text\n\nAnderson J, Wealleans J, Ray J: Endodontic applications of 3D printing. Int Endod J. 2018; 51(9): 1005–1018. PubMed Abstract \| Publisher Full Text\n\nKokol P: Smart dentistry. 2019. http://www.doi.org/10.17605/OSF.IO/UJRKT" }	[ { "id": "47891", "date": "29 May 2019", "name": "Wan Zaripah Wan Bakar", "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 article entitled “How ‘smart’ is smart dentistry?” is an interesting topic. It looks upon smart dentistry which is an important area especially as we are currently in the Fourth Industrial Revolution, where everything is changing fast with new technologies. It is no doubt that smart dentistry already exists with more new advanced technology that has emerged to facilitate clinical works.\n\nHowever with this bibliometric mapping methods study, the included papers were not critically appraised and the details information were not described clearly. Limitations that band it from being smarter such as cost and resources should be elaborated. It just show the overall general trend where a proper conclusion cannot be made, which ends up with very a weak conclusion. Maybe it is better to conclude that there is an improvement in smart dentistry with year changes especially in developed country as the gold standard level is yet not known.\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? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] }, { "id": "49289", "date": "01 Jul 2019", "name": "Praveen Arany", "expertise": [ "Reviewer Expertise Dental research", "biomaterials", "advanced technologies", "biophotonics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 advances of biomedical technologies and biomaterials is bringing about staggering improvements in clinical care, especially in dentistry that has always been at the forefront of adapting and utilizing the latest scientific advances. The authors do a good job of compiling the literature on this topic. This is a timely article as there is much excitement of how the rapid pace of technological advancements that are improving routine patient care.\n\nHowever, there seems to be fundamental confusion on what exactly constitutes a ‘Smart’ approach or technology. While digital impression, robotic surgery or precision additive (3D printing) manufacturing are phenomenal advances that are enabling individualized care, the techniques by themselves are not, in my personal opinion, a smart approach.\n\nThe following are examples of what may not be considered ‘Smart’ based on their ability to simply perform an enhanced passive or active function.\n\nPrecision-designed implant surface is NOT considered smart because it is passively driving a directed osseointegration response;\n\nDrug eluting biomaterial is NOT considered smart despite actively releasing an antimicrobial or disease modifying agent.\n\nThe classical definition, I believe, of a ‘Smart’ approach is a measured, controlled response to modulate a biological response based on some input/sensing. Some examples of Smart approaches are:\npH or enzyme activated payload release from biomaterials;\n\nSensors on appliances that enable (hardware/biomaterial) improved compliance or dose/treatment modulation;\n\nBig data (clinical, laboratory, epidemiological) and artificial intelligence enabling (software) improved precision and personalized care;\n\nOf the key topics/focus areas identified, the ‘smart’ aspect of each area could be specifically highlighted. For example, robotic surgery is a major technological advance but the use of diagnostic approaches, such as real-time radiographic or ultrasound imaging has enabled increased precision. Another key example in dentistry is the use of protease-sensitive dressings or materials that indicate active tissue turnover providing a visual cue for a clinical intervention.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\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/8-183
https://f1000research.com/articles/8-221/v1	26 Feb 19	{ "type": "Study Protocol", "title": "Methods of conduct and reporting of living systematic reviews: a protocol for a living methodological survey", "authors": [ "Assem M. Khamis", "Lara A. Kahale", "Hector Pardo-Hernandez", "Holger J. Schünemann", "Elie A. Akl", "Assem M. Khamis", "Lara A. Kahale", "Hector Pardo-Hernandez", "Holger J. Schünemann" ], "abstract": "Background: The living systematic review (LSR) is an emerging approach for improved evidence synthesis that uses continual updating to include relevant new evidence as soon as it is published. The objectives of this study are to: 1) assess the methods of conduct and reporting of living systematic reviews using a living study approach; and 2) describe the life cycle of living systematic reviews, i.e., describe the changes over time to their methods and findings. Methods: For objective 1, we will begin by conducting a cross-sectional survey and then update its findings every 6 months by including newly published LSRs. For objective 2, we will conduct a prospective longitudinal follow-up of the cohort of included LSRs. To identify LSRs, we will continually search the following electronic databases: Medline, EMBASE and the Cochrane library. We will also contact groups conducting LSRs to identify eligible studies that we might have missed. We will follow the standard systematic review methodology for study selection and data abstraction. For each LSR update, we will abstract information on the following: 1) general characteristics, 2) systematic review methodology, 3) living approach methodology, 4) results, and 5) editorial and publication processes. We will update the findings of both the surveys and the longitudinal follow-up of included LSRs every 6 months. In addition, we will identify articles addressing LSR methods to be included in an ‘LSR methods repository’. Conclusion: The proposed living methodological survey will allow us to monitor how the methods of conduct, and reporting as well as the findings of LSRs change over time. Ultimately this should help with ensuring the quality and transparency of LSRs.", "keywords": [ "living systematic review", "research methodology", "research reporting", "study protocol" ], "content": "Background\n\nThe living systematic review (LSR) is an emerging approach for evidence synthesis that uses continual updating to include relevant new evidence as soon as it is published1. LSR aims to make the relevant evidence available to users, soon after its publication. This could lead to “living knowledge translation” in the form of living guideline recommendations2 and living support systems to clinical and policy decisions3. LSRs are expected to take advantage of their currency to enhance the accuracy and utility of evidence synthesis3,4. Given their appeal, there appears to be an increase in the number of LSRs being conducted and published.\n\nConducting LSRs requires many of the steps of conducting traditional systematic reviews. However, they are more likely than traditional reviews to benefit from enabling technologies, including but not limited to automatic retrieval of full-text papers or for machine learning-assisted risk of bias assessment5. In addition, LSRs require steps that are specific to the living approach, such as frequent searches or protocols for triggering meta-analyses updating6.\n\nReporting LSRs should in principle adhere to all the elements of traditional systematic reviews, as detailed in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement7. However, LSRs’ protocols and final reports should also reflect the methodological features specific to their living approach. For example, LSR reports should highlight the rationale for choosing a living approach over a traditional approach. In addition LSR reports should describe the planned frequency of updating1, the editorial process, and the transition from a traditional to a living systematic review, if applicable1.\n\nWhile this is still an emerging field, we are not aware of any systematic assessments of the methodological approaches and reporting practices for LSRs. Such assessment would help with better understanding of the conduct and reporting of LSRs and in improving and standardizing them. That would ultimately help with ensuring the quality and transparency of LSR.\n\n\nStudy objectives\n\nThe objectives of this study are to:\n\n1. Assess the methods of conduct and reporting of living systematic reviews;\n\n2. Describe the life cycle of living systematic reviews, i.e., describe the changes over time to the methods and findings of living systematic reviews.\n\n\nMethods\n\nWe had defined living systematic reviews (LSR) as: “a systematic review that is continually updated, incorporating relevant new evidence as it becomes available.”1\n\nWe distinguish between the base LSR (which refers to the first published version of the LSR) and the subsequent LSR updates.\n\nThe operational criterion defining the eligibility of a LSR will be as follows: authors label their study as a ‘living systematic review’ (using this or similar terminology).\n\nWe will consider that an LSR lost its living status when its authors report it as such or when they fail to publish an update after a period that is triple that of the planned updates, according to the LSR protocol.\n\nWe define a ‘living methodological survey’ (LMS) as a study examining a specific aspect of research methodology (e.g., conducting or reporting of studies), with the findings of this survey being continually updated, incorporating relevant new data as they become available. The aim of a LMS is to reflect the current status of the research methodology aspect being assessed. The study involves no human subjects and requires no ethical approval.\n\nWe will conduct a LMS of LSRs. To address the first objective (i.e., assessing the methods of conduct and reporting of LSRs), we will first conduct a cross-sectional survey of studies that used LSR methodology (the ‘base LMS’). Then, we will update the cross-sectional surveys at regular time intervals (e.g., 6 months) by including LSRs published since the previous update (the ‘LMS updates’). The LMS update will exclude from its analysis a previously included LSR that loses its living status.\n\nTo address the second objective (i.e., describing the changes over time to the methods and findings of LSRs), we will conduct a prospective longitudinal follow-up of the cohort of all LSRs identified by the first study. The aim will be to describe the changes over time of the methods and findings of included published LSRs (e.g., frequency of updating, cessation of the living approach, adaptation to newly emerging technologies).\n\nWe will include studies labeled as ‘living systematic reviews’ addressing a health topic, irrespective of the health field (i.e., basic sciences, clinical, public health, health policy and systems), date or language of publication. We will include both ongoing LSRs and LSR protocols. We will also include studies addressing LSR methods and include them in an ‘LSR methods repository’.\n\nWe will search the following electronic databases: Medline, EMBASE and the Cochrane library. The search strategy uses both key words and MeSH terms judged to be relevant to our topic (Extended data 1). We developed our search strategy with the help of a librarian experienced in systematic review methodology. We used studies identified by a pilot search to refine the search strategy. We will set the alerts in the databases to search for newly published LSR and will follow for update(s) of the already included LSR. We will also search for LSRs in the Cochrane Library, the Epistemonikos database, as well as journals known (or found through this study) to publish LSRs. Lastly, we will contact groups conducting LSRs to identify eligible that we might have missed.\n\nReviewers will complete calibration exercises and then screen in duplicate and independently the titles and abstracts of citations identified by the search. We will obtain the full texts of any citations judged as potentially eligible by at least one reviewer. Reviewers will subsequently screen in duplicate and independently the full texts. They will check agreement and resolve any disagreements by discussion and involve a third review author as needed. We will record reasons for exclusion and summarize the results of the selection process using a PRISMA flow diagram. We will repeat this process for each LMS update.\n\nWe developed and pilot-tested a standardized data extraction form with detailed instructions (Extended data 2). Reviewers will complete calibration exercises and then extract data in duplicate and independently. They will compare results and resolve disagreements through discussion, or with the help of a third reviewer as needed. We will collect and manage study data using Research Electronic Data Capture (REDCap) tool hosted at the American University of Beirut. REDCap is a secure, web-based application designed to support data capture for research studies. We will export abstracted data from REDCap for every update our analysis.\n\nFor each included LSR, we will abstract information for each update on the following:\n\n1. General characteristics (Table 1):\n\nIf the publication is a LSR protocol, base LSR or LSR update (and its number);\n\nProtocol: if referred to, registered, published; or modified;\n\nType of field: basic sciences, clinical, health systems and policy, public health;\n\nDate of publication (year and month);\n\nWhether it is a Cochrane review\n\nNumber of authors (total, newly added, newly removed);\n\nWhether or not authors changed from previous version in terms of ranking/role (first author, last author, and corresponding author);\n\nFunding (type of funding: continuing vs. expired vs. new funding; source of funding; reporting on the role of funder);\n\nIf and how conflicts of interest were reported.\n\nIQR – interquartile range\n\n2. Systematic review methodology (Table 2, Table 3 & Table 4):\n\nIf the LSR builds on a previously published traditional SR;\n\nType of eligible primary studies (e.g., trials, non-randomized studies);\n\nRating certainty\n\nSummary of findings (SoF) tables\n\nTools and/or platform used for SR and output authoring (e.g., RevMan, GRADEpro);\n\nUse of task sharing processes (e.g., crowd participation);\n\nUse of machine assisted production processes (e.g., Cochrane RCT classifier);\n\nThe SR includes network meta-analysis;\n\nQuality of reporting, using the PRISMA checklist7;\n\nQuality of conduct, using the AMSTAR 2 tool8.\n\nLSR – living systematic review, SR – systematic review, SoF – summary of findings, RCT – randomized clinical trial\n\nLSR – living systematic review\n\nLSR – living systematic review\n\n3. Living approach methodology (Table 5):\n\nRationale for LSR provided (priority, uncertainty, emerging evidence, other);\n\nMethod of literature surveillance; sources (newly added, newly removed, retained); use of auto alerts; use of traditional search updates; search frequency, modification of search terms;\n\nUse of meta-analytic methods to adjust for frequent updating (e.g. trial sequential analysis, sequential meta-analysis, the Shuster method, Law of the iterated logarithm);\n\nChanges in LSR methodology compared to the previous version of the LSR.\n\nLSR – living systematic review, IQR – interquartile range\n\n4. LSR results (Table 6):\n\nElements of the PICO question modified;\n\nNumber of the LSR version;\n\nTime since preceding update;\n\nNumber of citations screened for the LSR update period;\n\nNumber of identified newly published eligible primary study protocols;\n\nNumber of identified newly published eligible primary studies;\n\nDealing with identified newly published eligible primary studies (i.e., incorporated or not);\n\nChange in statistical results, change in certainty of evidence, and change in conclusions.\n\nPICO – patient intervention comparison outcome, IQR – interquartile range\n\n5. Editorial and publication processes (Table 7):\n\nWhether the LSR version has been peer reviewed or not;\n\nWhether the managing editor and the peer reviewers are the same as for the previous version;\n\nTime required for the editorial and peer review processes;\n\nJournal (or platform) of publication; its impact factor; whether open access; whether it provides instructions for reporting of systematic reviews, and for LSRs respectively;\n\nWhether the journal (or platform) accommodates iterative versions of the same document (e.g., nano-publication approach, sub-doi);\n\nApproach to flagging changes in methods and findings for reader (new evidence).\n\nLSR – living systematic review, IQR – interquartile range\n\nWe will perform separate data analyses for two objectives included in this LMS (Figure 1):\n\n1. Cross sectional survey: For each LMS update, we will run a summary descriptive analysis of the variables of interest (related to conduct and reporting of LSRs) across the latest versions of included LSRs. With each LMS update, we will update the summary descriptive analysis, and archive the results of the previous LMS update. We will exclude from the cross sectional surveys the reviews that have lost their living mode status. We will run an analysis and present a graphical presentation of selected variables over time to show trends of changes in all LSRs in our survey updates, which adds a longitudinal dimension to this objective.\n\n2. Longitudinal follow-up: For each LSR, we will analyze the changes over time (i.e., the LSR lifetime) of selected variables (related to methods and results). We graphically present the findings to show their time trends.\n\n\nDissemination\n\nWe will publish the study protocol and the living methodological survey (LMS) in F1000Research journal. F1000Research journal has a dynamic publication process that allows adding versions of the LMS that represent the six-monthly updates, while making copies of the previous updates available.\n\n\nStudy status\n\nWe have finalized the search strategies for Medline, EMBASE, and the Cochrane library. We have drafted the data abstraction form. We are planning to launch the study after the protocol is published.\n\n\nDiscussion\n\nThe main objectives of this LMS are to assess the methods of conduct and reporting of LSRs and describe the changes over time to their methods and findings (the life cycle of living systematic reviews). This is the first methodological study that follows a living approach and that continuously surveys the methods of conduct, and reporting of LSRs. We aim to add newly published LSRs soon after their publication. This will ensure that our findings will be both current and representative of published LSRs.\n\nWe foresee that our LMS may be limited by the fact that we will focus on ongoing or published LSRs. As such, we may miss newly developed LSR methodologies that are being tested but not reported as of yet. We hope to overcome this shortcoming by surveying Cochrane groups and authors of LSRs on their unpublished LSR initiatives. Similarly, we might not obtain needed information from previous versions of published LSRs (i.e., when we run our first search) and therefore be unable to capture previous methodological approaches. Lastly, we might face some challenges in analyzing and presenting the time trends of our findings, since we expect heterogeneity in the field given the novelty of the approach. Maintaining our LMS in the living mode will require sustained efforts and resources.\n\nThe proposed LMS will allow us to monitor how the methods of conduct, and reporting as well as the findings of LSRs will change over time. In addition, we will be able to pinpoint potential gaps and research needs in the field of LSRs. We hope this survey will advance the methodology and subsequently the quality of LSRs, fostering in turn the currency of evidence supporting decision making for practice and policies.\n\n\nData availability\n\nNo data is associated with this article.\n\nFigshare: Extended data 1. Search strategies, https://doi.org/10.6084/m9.figshare.76880369\n\nFigshare: Extended data 2. LSR_data abstraction form_20190129.xlsx, https://doi.org/10.6084/m9.figshare.768782310\n\nData are available under a Creative Commons Attribution 4.0 International (CC BY 4.0) license", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work\n\n\nReferences\n\nElliott JH, Synnot A, Turner T, et al.: Living systematic review: 1. Introduction-the why, what, when, and how. J Clin Epidemiol. 2017; 91: 23–30. PubMed Abstract \| Publisher Full Text\n\nAkl EA, Meerpohl JJ, Elliott J, et al.: Living systematic reviews: 4. Living guideline recommendations. J Clin Epidemiol. 2017; 91: 47–53. PubMed Abstract \| Publisher Full Text\n\nElliott JH, Turner T, Clavisi O, et al.: Living systematic reviews: an emerging opportunity to narrow the evidence-practice gap. PLoS Med. 2014; 11(2): e1001603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMetzendorf MI, Featherstone RM: Ensuring quality as the basis of evidence synthesis: leveraging information specialists' knowledge, skills, and expertise. Cochrane Database Syst Rev. 2018; 4(9): ED000125. PubMed Abstract \| Publisher Full Text\n\nThomas J, Noel-Storr A, Marshall I, et al.: Living systematic reviews: 2. Combining human and machine effort. J Clin Epidemiol. 2017; 91: 31–37. PubMed Abstract \| Publisher Full Text\n\nSimmonds M, Salanti G, McKenzie J, et al.: Living systematic reviews: 3. Statistical methods for updating meta-analyses. J Clin Epidemiol. 2017; 91: 38–46. PubMed Abstract \| Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med. 2009; 151(4): 264–269. PubMed Abstract \| Publisher Full Text\n\nShea BJ, Reeves BC, Wells G, et al.: AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. BMJ. 2017; 358: j4008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhamis AM, Kahale LA, Pardo-Hernandez H, et al.: Search strategies. figshare. 2019. Paper. http://www.doi.org/10.6084/m9.figshare.7688036.v1\n\nKhamis AM, Kahale LA, Pardo-Hernandez H, et al.: LSR_data abstraction form_20190129.xlsx. figshare. 2019. Dataset. http://www.doi.org/10.6084/m9.figshare.7687823.v2" }	[ { "id": "48749", "date": "22 May 2019", "name": "Laurence Le Cleach", "expertise": [ "Reviewer Expertise Systematic review and meta-analysis in dermatology and living systematic review." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of this protocol is interesting and important. LSR is an emerging method and this study will contribute to enhance its methodology and publication process.\nMajor remarks The background could be developed in order to describe more precisely the issues, methodological difficulties, funding and publication of the LSR. When the first one was published? How many are currently published, are they published in scientific journals? For these last questions it is one of the aims of the study but authors should have an idea at least in the Cochrane library. In the discussion, other goals could be described such as contributing to an extension of PRISMA for LSR, providing information to contribute to build another editorial and publication process for living SR? Build collaboration with teams identified to collectively share tools, skills or enhanced methodology?\nMinor remarks\nThe author stated \"a librarian experienced in systematic review methodology\". Could they provide his/her name and affiliation. The authors stated in Abstract \"We will also contact groups conducting LSRs to identify eligible studies that we might have missed\" and in Methods: \"Lastly, we will contact groups conducting LSRs to identify eligible that we might have missed\". Indeed, In SR methodology it is normal to contact experts in the field and authors to obtain additional studies potentially missed. But in this setting is it relevant? The authors stated \"Then, we will update the cross-sectional surveys at regular time intervals (e.g., 6 months) by including LSRs published since the previous update (the ‘LMS updates’)\". Does this mean that you have not decided yet the frequency of your updates? Could you discuss that considering the methods and tools used if you are focused on RCT Living SR? Will this protocol be registered in Prospero as well? Could you explain why you chose to use AMSTAR rather than ROBIS? Could you explain how do you plan to published updates each 6 months?\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": "48191", "date": "07 Jun 2019", "name": "Terri Pigott", "expertise": [ "Reviewer Expertise Methods for meta-analysis and systematic review." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis protocol describes methods for the conduct of a living systematic review focused on the methods for living systematic reviews. The objectives of the study will be to assess the methods of conduct and reporting of living systematic reviews and to describe changes over time to living systematic review methods and findings. The research will include a cross-sectional survey of the methods and findings of living systematic reviews that will be updated every six months to include newly published living systematic reviews. The research will also analyze the changes over time in the methods and results used in the included living systematic reviews.\nThe study will make an important contribution. When new methods such as those for living systematic reviews are introduced in the literature, researchers wanting to use the method may have difficulty accessing articles and guidance. A living systematic review that documents methods for these types of systematic reviews gives researchers access to the most current applications of this method. These methods are also developing rapidly in the literature and living systematic reviews serve to keep the field aware of how researchers are applying the methods. As described in the protocol, living systematic reviews are more likely to use emerging technologies in the field such as machine learning-assisted methods. A living systematic review demonstrating the use of these emerging technologies is another potential contribution of the research.\nThe study methods are appropriate and use best practice standards for systematic review. The protocol does not provide a sense of the number of these reviews that are currently being conducted so it is not clear how large a review will result from this research. Sufficient details are provided to allow replication by other researchers.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/8-221
https://f1000research.com/articles/8-1093/v1	15 Jul 19	{ "type": "Research Article", "title": "Understanding the contribution of UK public health research to clinical guidelines: a bibliometric analysis", "authors": [ "Susan Guthrie", "Gavin Cochrane", "Advait Deshpande", "Benoit Macaluso", "Vincent Larivière", "Gavin Cochrane", "Advait Deshpande", "Benoit Macaluso", "Vincent Larivière" ], "abstract": "Background: There is an increasing need to understand the wider impacts of research on society and the economy. For health research, a key focus is understanding the impact of research on practice and ultimately on patient outcomes. This can be challenging to measure, but one useful proxy for changes in practice is impact on guidelines. Methods: The aim of this study is to map the contribution of UK research and UK research funders to the National Institute for Health and Clinical Excellence (NICE) public health guidelines, understanding areas of strengths and weakness and the level of collaboration and coordination across countries and between funders. The work consisted of two main elements: analysis of the references cited on NICE guidelines and interviews with experts in public health. Results: Across the papers cited on 62 NICE public health guidelines, we find that 28% of the papers matched include at least one UK affiliation, which is relatively high when compared to other health fields. In total, 165 unique funders were identified with more than three acknowledgements, based in 20 countries. 68% of papers which acknowledge funding cite at least one UK funder, and NIHR is the most highly cited funder in the sample.\n\nConclusions: The UK makes an important contribution to public health research cited on NICE PH guidelines, although the research does not appear to be bibliometrically distinct from other research sectors, other than having a relatively low level of international collaboration. However, the extent to which NICE public health guidelines reflect practice at the local authority level is less clear. More research is needed to understand the sources of evidence to support public health decision making at the local level and how NICE guidance can be made more applicable, timely and accessible in this new context.", "keywords": [ "Public health", "bibliometrics", "guidelines", "research funding", "UK" ], "content": "Introduction\n\nThere is an increasing need to understand the wider impacts of research on society and the economy. In the UK, this is reflected in the introduction of impact case studies into the national assessment system, the Research Excellence Framework, through which core funding is allocated to universities. More widely, there are moves to understand and demonstrate the impact of research outside the academic sphere. This reflects the wider political climate. Economic austerity and increased demands on public expenditure to provide direct services put pressure on research budgets and there is an increased need to show why investment in research is worthwhile.\n\nFor health research, a key focus is understanding the impact of research on practice and ultimately on patient outcomes. This can be challenging to measure, but one useful proxy for changes in practice is impact on guidelines. In the UK in particular, evidence that influences NICE guidelines can make a strong case for an impact on patient treatment in the NHS. This is an approach that has been used previously to analyse the way research affects practice in a number of fields1–3. However, the picture for public health is more nuanced, partly since interventions may often not be delivered through the NHS. In this study, we explore the impact of UK public health research on NICE public health guidelines.\n\nThe aims of this article are: to understand the importance of UK research and UK research funders in developing the evidence for the guidance provided, relative to other fields and across areas of public health; to compare the body of evidence that underpins NICE guidelines in this field to other areas; to consider the appropriateness of this approach in the public health context; and to consider the extent and way in which guidelines shape practice in this field.\n\n\nMethods\n\nThe aim of this study is to map the contribution of UK research and UK research funders to NICE public health guidelines, understanding areas of strength of weakness and the level of collaboration and coordination across countries and between funders. The work consisted of two main elements: analysis of the references cited on NICE guidelines and interviews with experts in public health. We discuss each of these methods in more detail in the remainder of this section.\n\nWe identified 62 currently active NICE public health guidelines from the NICE website (data extracted on 26/09/2016 ). The guidelines included are listed in Table 1. These guidelines were grouped into ten field categories, as indicated in Table 1, by two members of the team (GC, SG) based primarily on their titles, though a brief review of content was also conducted where necessary (e.g. if the title was ambiguous).\n\nThe references were extracted from the guidelines manually into a database, producing a total of 31,409 references across all guidelines. It should be noted that many of these guidelines do not include all or, in some cases, any of the references for the evidence that underpinned their development directly. Often, separate evidence summaries are provided (also on the NICE website) for the guidelines – sometimes several for one guideline – which provide references for the underpinning evidence. We extracted the references from these evidence summaries as well as directly from the guidelines themselves.\n\nThe references were matched in the Web of Science (WoS) database. Once duplicates were removed and documents which were not journal articles excluded, we were able to produce a set of 12,706 papers from the guidelines matched in Web of Science. This is a low proportion of the total references extracted, but reflects a high level of policy documents and other non-journal material referenced in the guidelines.\n\nFor each of these papers matched in WoS, we were able to analyse the institutional affiliations of the contributing authors, based on the address information provided. We used this to look at the countries in which the research was conducted. We used a full counting approach, to allow for comparisons with previous studies in the area.\n\nWe also have the date of publication of each of the articles matched in WoS. By comparing this to the publication date of the relevant guideline, we are able to produce an estimate of the ‘knowledge cycle time’ – that is, the time between the publication of a paper and its citation on the guideline.\n\nFinally, for a number of the publications, we were also able to extract the names of the funders supporting the research from the funding body acknowledgements (FBAs) on the publications. This is only available in WoS for publications from 2008, and even since 2008 the data is not complete, partly because authors do not always cite their funding sources completely and accurately (and sometimes not at all). These data were available for 15% of the papers matched in WoS.\n\nUsing these data sets we were able to analyse the contributions and levels of collaboration across funding bodies, countries and between institutions on the publications cited on public health guidelines, by area of public health and as a whole. Analysis was carried out using Microsoft Excel and R.\n\nInterviews were conducted with eleven stakeholders engaged in public health-related decision-making and public health research in the UK. Interviewees were selected to ensure a mix of geographical settings and experiences. At the time of the interview, seven of the interview participants were directors of public health at the local authority level, tasked with responsibility for delivering public health interventions in the UK since 20134. Four interview participants were either senior academics or researchers with significant experience of public health in the UK. In accordance with the terms of interview participation, any quotes or attributions have been anonymised where applicable.\n\nInterviews were typically 30–60 minutes long and were conducted by telephone in a semi-structured format, based on the interview protocol provided in Table 2. An anonymised list of interviewees, along with their designation and expertise, is included in Table 3. Interviews were recorded, subject to the consent of the individuals, and summary notes written up after each interview (not full transcripts). Interviews were conducted under the principle of informed consent with interviewees informed that the recordings and notes would only be used by the study team for the purposes of the study. These files are stored on secure servers and recordings will be destroyed one year after study completion. The interview notes were analysed, and key themes and common messages extracted to generate some of the insights and the underlying context described, taking a thematic analysis approach. The emergent themes from the interviews are covered in more detail in the Discussion section of this paper.\n\n\nEthics and consent\n\nThe need for ethical approval was waived for this study by the RAND Human Subjects Protection Committee since interviews were conducted by individuals in their professional capacity only. Oral consent was sought from interviewees to record the interviews, with the interview recording and accompanying notes/transcripts solely for the use of the study team. Oral, rather than written, consent was sought as interviews were conducted by telephone. All participants are anonymised in the discussion in the article and we have ensured all quotes used could not identify the contributor.\n\n\nResults\n\nIn this section, we discuss the findings from the bibliometric analysis of the citation data included in the NICE guidelines. We discuss this in two main sections: (1) contribution of the UK as a provider of research, through an analysis of the institutional affiliations associated with cited papers; (2) contribution of the UK as a funder of research through an analysis of the funding body acknowledgements associated with cited papers.\n\nThe first level of analysis was to assess the distribution and co-occurrence of institutional affiliations from publications cited in the guidelines. This allows us to look at the contribution of the UK and other countries to the research conducted that underpins this set of guidelines.\n\nWe found that 28% (2,627/9,391) of the papers matched include at least one UK affiliation. Of the 2,627 papers with UK institutions, 2,114 papers (80%) contained only UK based affiliations, suggesting the proportion of international collaboration to be around 20% with non-UK based institutions. This is below the general average for international collaboration in research across the UK5,6.\n\nWe can also look at the contribution from other countries. Across the 9,391 papers, we identified 5,617 institutions across 93 countries. Figure 1 shows the distribution of institutions affiliated with the papers.\n\nHowever, though many countries make some contribution, the majority of the research comes from a more limited set of countries. As shown in Figure 1, there are four countries (UK, US, Canada and Australia) who have contributed to over 500 papers, and collectively, these four countries have contributed to 90% of the papers. The largest contributor is the US - 47% of papers have a US institution affiliation (4,419). This is likely to reflect the scale of public health research conducted in the US, relative to other countries7,8.\n\nDividing the papers by subject area, we see that the contribution of UK research to the evidence used in guidelines is broadly similar for each subject area. Figure 2 shows the total number of papers for UK, US, Canada and Australia by public health subject area. The US accounts for the majority of papers in each subject area, with the exception of diabetes and oral health, where the UK accounts for the majority of papers (40% and 29% respectively). Papers from non-Anglophone countries account for less than 4% of papers in each subject area with the exception of: oral health, where 9% of papers are from Sweden; mental health, where 6% of papers are from the Netherlands; cardiovascular, where 4% of papers are from Finland.\n\nWe can also look at the contribution of specific institutions, which provides some scope to identify ‘centres of excellence’ in public health research. In the UK, 1,181 institutions were identified, featuring on 2,627 papers. Table 4 shows the top ten institutions from the UK in the dataset, also mapped in Figure 3.\n\nFigure 4 outlines the network of institutional affiliations on papers with a UK address. In the network, each node represents an institution and the connecting lines indicate these institutions’ co-acknowledgement on papers (a thicker line indicates a greater number of co-acknowledgements). The size of a node is proportional to the number of papers with a UK corresponding address affiliated to the institution. Institutions with fewer than 4 papers with an associated UK address are not included in the network. Nodes are coloured by the location of the institutions.\n\nEach node represents an institution and the connecting lines indicate these institutions’ co-acknowledgement on papers (a thicker line indicates a greater number of co-acknowledgements). The size of a node is proportional to the number of papers with a UK corresponding address affiliated to the institution. Institutions with fewer than 4 papers with an associated UK address are not included in the network. Nodes are coloured by the location of the institutions.\n\nAside from the top UK institutions in the network (see Table 4), institutions from other Anglophone countries, such as Australia, US, New Zealand and Canada, tend to be the most prominent non-UK institutions in the network.\n\nOverall, the UK is an important contributor of research to the evidence base supporting NICE public health guidelines, contributing to 28% of papers, alongside other Anglophone countries. This may partly reflect biases in the WoS towards English language publications but may also reflect the strength of these countries in the field. Clarke et al.’s bibliometric overview of public health research in Europe7 found that only 3.5% of research papers were published in non-English languages. This does not differ significantly by field of research within public health.\n\nThe second level of analysis was to assess the distribution and co-occurrence of recorded funding body acknowledgements (FBAs) from publications cited in the guidelines. This bottom-up approach has been used in various studies to map the funding landscape of a particular field, explore interactions among research funders and assess trends in research funding9–12.\n\n1,420 papers contain FBAs with an average of 2.98 acknowledgements per paper. This represents 15% of the dataset, which is lower than other studies12 but may reflect the fact that 67% of the papers in the dataset were published before 2008 – the year WoS began collecting data on FBAs.\n\nAcross these papers, 165 unique funders were identified with more than three acknowledgements, based in 20 countries. Table 5 shows the total number of papers and FBAs by funder location.\n\nWhile there are significantly more US-based institutions affiliated with papers in the dataset, there are more papers with UK-based funders acknowledged than the US. NIHR is the most acknowledged funder in the dataset, accounting for 11% of papers with FBAs. Taken together with the UK Department of Health, it accounts for 226 papers (16% of papers with FBAs). Table 5 shows the top ten most acknowledged funders in the dataset, which account for 60% of the papers with FBAs.\n\nAlong with the UK Department of Health, research councils (such as MRC and ESRC) and disease-specific charities working in public health (such as British Heart Foundation and Cancer Research UK) are the most prominent UK funders in the dataset. In addition to UK funders, we see two US funders and one Australian funder in the top ten.\n\nGovernment funders make up the majority of the 165 funders in our data set, accounting for eight of the top ten funders and around 72% of all funding acknowledgements (Figure 5). While our previous study on the FBAs associated with global mental health research12 found a similar distribution in the overall number of acknowledgements (with government funding accounting for 68%), the study found a significantly larger proportion of charities, foundations and non-profits in relation to the total number of funders.\n\nFigure 6 outlines the network of funders acknowledged in papers across the dataset. In the network, each node represents a funder and the connecting lines indicate these funders’ co-acknowledgement on papers (a thicker line indicates a greater number of co-acknowledgements). The size of a node is proportional to the number of papers in the network. Funders with fewer than ten papers are not included in the network. Nodes are coloured by the location of the funder.\n\nEach node represents a funder and the connecting lines indicate these funders’ co-acknowledgement on papers (a thicker line indicates a greater number of co-acknowledgements). The size of a node is proportional to the number of papers in the network. Funders with fewer than 10 ten papers are not included in the network. Nodes are coloured by the location of the funder.\n\nThe most prominent funders in the network are the most frequently-acknowledged US and UK funders (see Table 5). There is also a relatively large contingent from Australia, multilateral funders such as the EU and the WHO, as well as a number of pharmaceutical companies, such as Pfizer, Merck, GSK and Sanofi. Government funders in Canada, the Netherlands and Scandinavia are also visible.\n\nOf the 165 unique funders identified with more than three acknowledgements, 43 funders (26%) are based in the UK. We have split UK government funding, which make up 42% of the UK funders, into five funder groups for the purposes of analysis.\n\nThe “UK Department of Health”, which includes National Institute for Health Research (NIHR) and NHS England as well as the UK Clinical Research Collaboration (UK CRC) and National Institute for Health and Clinical Excellence (NICE);\n\nResearch Councils, primarily the UK Medical Research Council (MRC), make up the majority of funding acknowledgements in the dataset. Additional research councils in the data set includes: Economic and Social Research Council (ESRC), Biotechnology and Biological Sciences Research Council (BBSRC), Engineering and Physical Sciences Research Council (EPSRC), and Arts and Humanities Research Council (AHRC);\n\nDevolved government departments, in Scotland, Wales and Northern Ireland;\n\nNon-health focussed central government departments, such as the Department for International Development (DFID);\n\nNon-departmental public bodies, both with a health focus (e.g. British Health and Safety Executive) and non-health focus (e.g. UK Food Standards Agency, Higher Education Funding Council for England (HEFCE)).\n\nFigure 7 outlines the network of funders acknowledged on papers with an associated UK address. In the network, each node represents a funder and the connecting lines indicate these funders’ co-acknowledgement on papers (a thicker line indicates a greater number of co-acknowledgements). The size of a node is proportional to the number of papers in the network. Funders with fewer than 3 papers with an associated UK address are not included in the network. Nodes are coloured by the location of the funder.\n\nEach node represents a funder and the connecting lines indicate these funders’ co-acknowledgement on papers (a thicker line indicates a greater number of co-acknowledgements). The size of a node is proportional to the number of papers in the network. Funders with fewer than 3 papers with an associated UK address are not included in the network. Nodes are coloured by the location of the funder.\n\nBreaking this down by subject area, we see that the contribution of research funders varies according to the subject area of the guideline. Figure 8 highlights the number of papers cited on the four main subject areas of NICE public health guidelines across the top ten research funders identified. For guidelines on addiction and substance abuse (of which the majority are primarily focussed on smoking), the US National Institutes of Health is acknowledged the most, followed by Cancer Research UK and NIHR. The US National Institutes of Health is also the most frequently acknowledged funder on mental health and wellbeing guidelines. However, for guidelines focussed on obesity and nutrition, the Australian National Health and Medical Research Council (NHMRC) is the most frequently acknowledged funder and for guidelines on physical activity, NIHR is the most acknowledged funder, with the top four funders all based in the UK. However, it is important to reiterate in interpreting this data that only 15% of papers in the dataset contain FBAs.\n\nNCI, National Cancer Institute (US); CIHR, Canadian Institutes of Health Research; CRUK, Cancer Research UK; ESRC, Economic and Social Research Council (UK); BHF, British Heart Foundation; NHMRC, National Health and Medical Research Council (Australia); DH, Department of Health (England); MRC, Medical Research Council (UK); NIH, National Institutes of Health (US); NIHR – National Institute for Health Research (England).\n\n\nDiscussion\n\nBased on the analysis above, we can conclude that UK public health research and research funders make an important contribution to NICE guidelines. Of the publications matched in Web of Science, 28% have at least one UK author, which is relatively high compared to other fields where such analysis has been conducted, as shown in Table 6, suggesting that context is important to the application of research to policy in this field. In addition, 68% of papers which acknowledge funding cite at least one UK funder, and NIHR is the most highly cited funder in the sample. This suggests that UK funded research in particular is important here, perhaps since research priorities are likely to correspond to policy and practice needs in the UK context. However, there may be other reasons for this – for example, it may be that UK funders are more likely to require acknowledgement of their funding on publications, or that the more recent papers, which are more likely to have funding acknowledgements, are more likely to be from UK sources. This may make sense, as this may correspond to more recent research that reflects the implementation of research in practice, building on a wider body of research which could come from a wider range of sources and countries, since the contextual factors are less crucial to the relevance of this broader underpinning body of knowledge.\n\nAlthough we can make the case that UK public health research does make an important contribution to NICE guidelines, the extent to which this reflects practice, and hence the extent to which UK public health research can be said to be influencing practice in the UK, is less clear. Based on the interviews we conducted, the extent to which NICE guidelines are seen to reflect practice varies significantly, depending on the prevailing circumstances of each local authority area. NICE guidelines on some aspects, such as obesity, smoking cessation, children’s health and cancer prevention, are considered relevant across the board. However, due to budgetary reasons, statutory requirements and geographical variations, local practice can differ significantly (INT01; INT03; INT04; INT08; INT10; INT11).\n\nSince the politics of representation and the principle of democratic accountability are crucial to the functioning of local authorities, NICE guidelines, although considered, are not always the definitive evidence for commissioning or recommissioning services (INT02; INT08; INT10). The extent to which the guidelines are deemed to reflect practice also depends on the commissioning / recommissioning cycles. Multiple interviewees argued that NICE guidelines can be considered crucial in relation to evaluating existing services and whether the services meet the requirements set by the guidelines (specifically if budget cuts are to be achieved) (INT01; INT03; INT05; INT11).\n\nDepending on how the local authorities understand and interpret the role of NICE guidelines, the perceptions of the extent to which NICE guidelines reflect practice varies. Multiple interviewees highlighted the need to communicate more clearly that NICE guidelines are only meant to help make a public health-related decision and not present a ready-made decision on the behalf of the authorities (INT02; INT03; INT04; INT05; INT08; INT10). These interviewees contended that NICE guidelines are aimed at achieving national relevance and the extent to which some of the guidance can be implemented in the context of specific local challenges would need to vary accordingly. This is reflected in how local authorities with predominantly rural and semi-urban areas with sparse populations are likely to diverge in their approach when considering the guidelines to be implemented (INT04; INT05; INT06; INT08). The following quote elaborates on how the needs of rural, semi-urban areas differ when compared to urban areas. The quote has been paraphrased to protect the anonymity of the interview participant:\n\n“… It’s a common problem [the urban focus] that we are discussing with Public Health England. Identifying the requirements for rural community [is a challenge]. If you look at sexual health guidelines (HIV, syphilis etc.), the outreach worker in rural areas sometimes has to travel 5 times the distance than the one in central London. How do you adapt service models to that? Some of the NICE guidance refers to smartphone apps and relies on people having access to the Internet, whereas in the rural areas, superfast broadband is non-existent. ” [INT04]\n\nGiven the constraints of budget cuts / austerity, more than one director of public health mentioned that they sought evidence from other (non-UK) countries in relation to their decision-making for local public health priorities (INT04; INT06). Since there is insufficient precedent for the use of such evidence, the interviewees also suggested that guidance on weighing the appropriateness of the evidence for a particular local authority context would be useful. Such guidance was perceived as being potentially useful to practice and decision-making at local authority area. The way NICE guidelines are perceived to consider hierarchy of evidence also forms an important part of their use in practice (INT11). The type of evidence (based on Randomised Control Trials i.e. RCTs, modelling in some cases) varies and some interviewees suggested that as part of recommendations, the level of evidence is not always identified. This can pose challenges to local authorities when deciding their needs for evidence or additional evidence synthesis. For evidence deemed high-level and not necessarily based on detailed case studies, the NICE guidelines were often considered to be less reflective of the actual practice by some of the interviewees (INT01; INT03; INT04; INT11).\n\nAn important document at the local authority level is the health and wellbeing strategy. In recognising that NICE guidelines focus on treatment and prevention measures, local authorities emphasise engagement with local stakeholders and locally produced intelligence when creating and updating health and wellbeing strategies. Given that the local authorities are best placed to gather and act upon local intelligence, multiple interviewees suggested that the upstream for local authorities into planning and regulation related to public health decision-making needs further consideration. According to some interviewees, such upstream input could strengthen the guidelines and their use in practice (INT03; INT04; INT05). The following quote provides further insight into this position:\n\n“We have an input into the development of national policy. I am not sure how much it determines what national do, because their work is more centralised. You do find situations when national policy and recommendations differs from the challenges at the local level authority.\n\nWhat national does is great if it feeds into what happening locally. But differences can exist from what the national agenda suggests. I am not sure how much we influence the national agenda. We certainly try.” [INT05]\n\nAn alternative approach to look at the impact of research on practice in public health would be to analyse documentation at the local authority level. However, based on the interviews and desk research we conducted, there are likely to be many challenges with this alternative approach:\n\nThe local authorities follow different processes and approaches to document and store their approach and policies for public health-related decision-making. This makes it time-consuming to track and identify local authorities’ public health-related documentation.\n\nAlthough there is a degree of consistency in terms of storing documentation related to the Joint Strategic Needs Assessment (JSNAs), the practices in terms of citations and referencing vary significantly. with many documents setting out strategy including no formal referencing of research or underpinning evidence.\n\nIn addition to the different practices for referencing and citation of the evidence, the use of evidence at the local level (including local statistics, local survey data and local population data) varies significantly. The citation and referencing of local evidence in this form varies, which makes the analysis of local authorities’ documentation a time-consuming task.\n\nDocument sharing with local government associations varies and the processes can often be informal in nature, which makes it difficult to authoritatively establish the reach and impact of the documentation produced by the local authorities.\n\nDocument sharing between local authorities varies based on local areas and extent of coordination and leadership approaches are also different. This has an influence on the documentation publicly available and the manner in which evidence is cited.\n\nGiven the emphasis on evidence-based decision-making in case of some priorities, evidence gathered and analysed by the teams that support the DPH and the documentation is internally provided. This evidence may not be formally cited depending on the nature of documentation which may / may not be available publicly.\n\nThe public availability of the documents is shaped by contracting decisions and whether recommissioning is necessary in relation to a public health service. Consequently, making a realistic assessment of the evidence included in documentation related to public health was not feasible in the timescales allocated for this research.\n\nAs noted above, a key limitation of this analysis is the extent to which we can link citation on NICE guidelines to changes in practice. In some areas of health research there are reasonable grounds to assume that practice should, at least to some extent, reflect, or be moving towards, best practice, as set out in guidelines. However, this is less clear in public health, where implementation can take multiple routes and is very much local-context dependent. In addition, we can also add the usual caveats around the use of bibliometrics, some of which are clearly emphasised here. Firstly, only a relatively small proportion of the publications from the guidelines were matched in Web of Science, which may be because much of what is cited on guidelines are not journal publications. This means the analysis here is only a reflection of that portion of the evidence used to underpin guidelines. Secondly, there are a number of known biases in the coverage of bibliometric databases – notably in relation to language, where English language publications are more covered than other languages, which may bias towards publications from the UK and other Anglophone countries. Finally, it should be noted that a full counting approach is used in the analysis here, meaning we look at the number of papers to which researchers or funders from a particular country has contributed. For many of these, there will also be contributions from other countries, and this analysis cannot account for the extent of the contribution of the different authors and/or funders, which may differ substantially.\n\nIn terms of the interviews conducted, we are of course limited to the range of viewpoints provided by the set of individuals to whom we spoke. There may be perspectives which we did not capture, and there may be biases inherent of the sample of individuals willing to speak about their experience and knowledge of evidence supporting practice in public health. However, we conducted sufficient interviews that we reached ‘saturation point’, that is, additional interviews did not seem to be adding substantially to the range and nature of information captured. We also compared the interview findings to wider published evidence on the topic.\n\n\nConclusions\n\nUK public health research makes an important contribution towards NICE guidelines, accounting for 28% of all cited papers. Research is broadly collaborative and coordinated across funders within the UK and in this respect, public health as a research field does not appear to be bibliometrically distinct from other research sectors. As the largest health research funder in the UK, it is also not surprising that the NIHR is the most acknowledged funder on papers cited in NICE public health guidelines.\n\nThere is, however, a relatively low-level of international collaboration on UK papers cited in the guidelines. Given the nature of the dataset, this may be expected for two reasons. Firstly, 23% of the papers included in the guidelines were published before the year 2000, when levels of international collaboration were lower. Secondly, the interviews conducted with public health experts highlight the local nature of public health research, which may mean that international collaboration is less likely in these fields.\n\nHow far NICE public health guidelines reflect practice conducted at the local authority level is less clear. Therefore, the extent to which public health research truly drives practice is harder to determine. More research is needed to understand the sources of evidence to support public health decision making at the local level, and how NICE guidance can be made more applicable, timely and accessible in this new context.\n\n\nData availability\n\nInterviews were conducted under the principle of informed consent. Interview participants were informed that recordings, transcripts and notes from the interviews were solely for the use of the project team and therefore these cannot be made publicly available, even in an anonymised form. If researchers wish to access these notes for the purposes of further research, they may contact the corresponding study author, Dr Susan Guthrie, by email (sguthrie@rand.org), providing details of the information required and the intended use of the data. Subject to approval by the RAND Human Subjects Protection Committee, we will then contact the interview participant(s) in question to seek their permission to share the interview notes for the purpose specified.\n\nFigshare: Papers from NICE public health guidelines matched in Web of Science.csv. https://doi.org/10.6084/m9.figshare.8053187.v113\n\nThis project contains the following underlying data:\n\n- Papers from NICE public health guidelines matched in Web of Science.csv (List of papers cited on NICE public health guidelines which were matched in Web of Science)\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 is an independent study conducted by the PRiSM unit, commissioned and funded by the Policy Research Programme in the Department of Health.\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\nKryl D, Allen L, Dolby K, et al.: Tracking the impact of research on policy and practice: Investigating the feasibility of using citations in clinical guidelines for research evaluation. BMJ Open. 2012; 2(2): e000897. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlover M, Buxton M, Guthrie S, et al.: Estimating the returns to UK publicly funded cancer-related research in terms of the net value of improved health outcomes. BMC Med. 2014; 12(1): 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThelwall M, Maflahi N: Guideline references and academic citations as evidence of the clinical value of health research. J Assoc Inf Sci Technol. 2016; 67(4): 960–966. Publisher Full Text\n\nLocal Government Association: Public health in local government– one year on. 2014. Accessed 5 Oct 2018. Reference Source\n\nAdams J, Gurney KA: The implications of international research collaboration for UK universities. Digital Science. 2016; Accessed 5 Oct 2018. Reference Source\n\nWitze A: Research gets increasingly international. Nature. 2016; Accessed 5 Oct 2018. Publisher Full Text\n\nClarke A, Gatineau M, Grimaud O, et al.: A bibliometric overview of public health research in Europe. Eur J Public Health. 2007; 17(suppl 1): 43–9. PubMed Abstract \| Publisher Full Text\n\nParks, et al.: Sketching the landscape of public health research using bibliometric techniques, 2006–2015. In preparation.\n\nNHS Executive: Putting NHS Research on the Map: An Analysis of Scientific Publications in England, 1990-97. Wellcome Trust. 2001. Reference Source\n\nGarau M, Mordoh A, Sussex J: Exploring the Interdependency between Public and Charitable Medical Research. Office of Health Economics; Accessed 5 Oct 2018. 2011. Reference Source\n\nShah K, Sussex J, Hernandez-Villafuerte K, et al.: Exploring the Interdependencies of Research Funders in the UK. Project Report. Cancer Research UK. 2014; Accessed 5 Oct 2018. Publisher Full Text\n\nPollitt A, Cochrane G, Kirtley A, et al.: Project Ecosystem: Mapping the global mental health research funding system. Santa Monica, CA: RAND Corporation. 2016; Accessed 5 Oct 2018. Reference Source\n\nGuthrie S, Larivière V: Papers from NICE public health guidelines matched in Web of Science.csv. figshare. Dataset. 2019." }	[ { "id": "51248", "date": "17 Jul 2019", "name": "Mike Thelwall", "expertise": [ "Reviewer Expertise Bibliometrics" ], "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 descriptive analysis of references in UK NICE guidelines using bibliometrics together with stakeholder interviews for context. Although clinical guideline citations have been investigated before, I think this is the first to match citations with the web of science and to analyse them in conjunction with interviews. The paper contains a lot of results, is well written and clearly explained. The interviews do not seem to have been systematically analysed with a recognised qualitative method but I think this is fine given that they are used to support and follow up from the bibliometric analysis, as well as to address pre-defined questions. It also does not have a literature review component summarising what is known about NICE guidelines from previous research but it does not really need it.\nFor the sentence, “This is below the general average for international collaboration in research across the UK”, could you find from the Web of Science what the average is for public health? If not, the science-wide comparison is not very meaningful.\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": [] }, { "id": "77488", "date": "28 Jan 2021", "name": "Claudio Tresoldi", "expertise": [ "Reviewer Expertise Epidemiology", "Statistics" ], "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 unusual and addresses a particular topic, maybe more difficult for a reader outside UK, but it is however interesting and well done, presenting useful and remarkable results. However, some minor adjustments are recommended to improve it.\nSpecific points:\nAbstract\nThe acronyms NIHR and PH here are not defined.\nIntroduction\nThe acronym NHS is not defined.\nMethods - Analysis of references cited on NICE guidelines\nThere is no reference for software R; in scientific publications the following is usually used:\nR Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/\nResults - Contribution of the UK as a funder of public health research\nThe second reference to Table 5 (Page 8, line 21 of that paragraph: “Table 5 shows the top ten most acknowledged funders in the dataset, which account for 60% of the papers with FBAs”) seems not pertinent, as in Table 5 there is not a list of funders.\nDiscussion\nPage 14, penultimate line of left column: the acronym DPH is not defined.\nTables\nTable 2: there are acronyms used without any previous definition, i.e. LAs, CCGs, PH.\nFigures\nFigure 1: grey color is not defined in the legend; it is supposed to indicate countries counting zero contributing papers, but this is contradictory with the group represented by the lightest blue, which seems to include also the 0.\n\nFigure 4 is really too complex and hard to understand! Thicker lines are difficult to distinguish. Moreover, it is also cut at the top and the bottom.\n\nFigure 6 and 7 are less confuse than Figure 4, but thicker lines are still hard to distinguish.\n\nFigure 8: it is not clear the meaning of the numbers n in brackets after each of the four headings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1093
https://f1000research.com/articles/8-114/v1	29 Jan 19	{ "type": "Method Article", "title": "Updating the evidence on the effectiveness of the alcohol reduction app, Drink Less: using Bayes factors to analyse trial datasets supplemented with extended recruitment", "authors": [ "Claire Garnett", "Susan Michie", "Robert West", "Jamie Brown", "Susan Michie", "Robert West", "Jamie Brown" ], "abstract": "Background: A factorial experiment evaluating the Drink Less app found no clear evidence for main effects of enhanced versus minimal versions of five components but some evidence for an interaction effect. Bayes factors (BFs) showed the data to be insensitive. This study examined the use of BFs to update the evidence with further recruitment. Methods: A between-subject factorial experiment evaluated the main and two-way interaction effects of enhanced versus minimal version of five components of Drink Less. Participants were excessive drinkers, aged 18+, and living in the UK. After the required sample size was reached (n=672), additional data were collected for five months. Outcome measures were change in past week alcohol consumption and Alcohol Use Disorders Identification Test (AUDIT) score at one-month follow-up, amongst responders only. BFs (with a half-normal distribution) were calculated for those for which we had outcome data (BF<0.33 indicate evidence for null hypothesis; 0.33<BF<3 indicate data are insensitive). Results: Of the sample of 2586, 342 (13.2%) responded to follow-up. Data were mainly insensitive but tended to support there being no large main effects of the enhanced version of individual components on consumption (0.22<BF<0.83) or AUDIT score (0.14<BF<0.98). Data no longer supported there being two-way interaction effects. In an unplanned comparison, participants receiving the four most promising components averaged a numerically greater reduction in consumption than those not receiving any (21.6 versus 12.1 units), but the data were insensitive (BF=1.42). Conclusions: Data from extended recruitment in a factorial experiment evaluating components of the Drink Less app remained insensitive but tended towards individual and pairs of components not having a large effect. There was weak evidence for a synergistic effect of four components. In the event of uncertain results, calculating BFs can be used to update the strength of evidence of a dataset supplemented with extended recruitment.", "keywords": [ "Bayes Factors", "digital interventions", "alcohol reduction", "smartphone apps" ], "content": "Introduction\n\nA factorial experiment evaluating the effect of ‘enhanced’ versus ‘minimal’ versions of five components of the alcohol reduction app, Drink Less, found no clear evidence for simple effects but did find evidence that two-way combinations of certain ‘enhanced’ components together resulted in greater reductions than ‘minimal’ versions1. This was a planned analysis but should be interpreted with caution as the two-way interactive effects were not specifically hypothesised a priori and were part of multiple interactions tested. Findings of this sort are not uncommon in experimental studies. One approach is to start another randomised trial specifically to test this hypothesis. A potentially more efficient alternative is to extend the trial with further recruitment and test this and other hypotheses using Bayes factors2,3. We used this approach with the Drink Less app.\n\nBayes factors are a measure of strength of evidence and allow researchers to ‘top-up’ their results from one trial with additional data collected, regardless of the stopping rule, unlike frequentist statistics2. The use of Bayes factors supports efficient, incremental model building3, as evidence can be continuously accumulated until it is clear whether there is an association or not2,4. The rapid accumulation of large amounts of data about digital behaviour change interventions (DBCIs) offers the opportunity to apply emerging methods to their evaluation. DBCIs often have the capacity to continue automatic data collection beyond the end of a trial with little or no additional resources. This paper will illustrate how Bayes Factors can be used to optimise a DBCI by updating evidence from an effectiveness trial using the example of Drink Less—an alcohol reduction app.\n\nBayes factors are the ratio of the average likelihood of two competing hypotheses being correct given a set of data and can overcome some of the issues associated with traditional frequentist statistics5. They indicate the relevant strength of evidence for two hypotheses; when evaluating interventions, the two hypotheses are typically the alternative hypothesis (the intervention had the desired effect) and the null hypothesis (the intervention had no effect). Bayes factors, unlike frequentist statistics, can distinguish between two interpretations of a non-significant result: i) support for the null hypothesis of ‘no effect’ and ii) data are insensitive to detect an effect i.e. ‘unsure about the presence of an effect’5,6. Calculating Bayes factors to supplement frequentist statistics is a quick and simple procedure with several software packages freely available (e.g. an online calculator developed by Zoltan Dienes7). Researchers are actively encouraged to supplement, or even replace, classical frequentist hypothesis testing with a Bayesian approach to provide greater interpretative value to any non-significant results8. This is important as often non-significant results are misinterpreted as evidence for no effect; a review of trials conducted in addictions research found that the reporting of ‘no difference’ was only appropriate in a small number of papers reporting this 9.\n\nThe use of Bayes Factors also has another major advantage over the traditional frequentist approach that relates to the stopping rule. The traditional frequentist approach necessitates a strict stopping rule and a single analysis of data. Typically, this involves an a priori power calculation to specify the required sample size for data collection and the trial to end at that point. Subsequent ‘topping-up’ of existing data and re-analysing the new larger data set is ‘prohibited’10. This is because any p-value between 0 and 1 is equally likely if the null hypothesis is true, regardless of how much data are collected11. Therefore, given enough time and data collection, a significant p-value will always be obtained even if the null hypothesis is true10. So if researchers find a non-significant result—which cannot distinguish between support for the null hypothesis and being insensitive to detect an effect—then a new study would be required to build on these findings. Restarting the process is a waste of research resources but necessary in the context of using a frequentist approach for analysis because additional data collected cannot be analysed. However, this is not the case when using Bayes factors, as they are driven towards zero when the null hypothesis is true and additional data are collected10. Therefore, researchers may use Bayes factors to analyse additional data to complement an employed stopping rule2.\n\nIn the evaluation of DBCIs, using Bayes factors is beginning to complement traditional frequentist statistics4,12, and analysing additional data would be of particular benefit. Data collection for a DBCI effectiveness trial is typically automated and therefore does not require additional resources to continue after a pre-specified sample size is reached. Rapid evaluations of DBCIs and efficient accumulation of evidence can be used to inform future versions, keeping pace with advances in technology. Using Bayes factors to update findings about the relative plausibility of the two hypotheses allows researchers to assess the DBCI’s effectiveness in an ongoing manner4. This remains useful when deciding about whether there is sufficient evidence to demonstrate effectiveness and, therefore, continued development13. To the authors’ knowledge, no DBCIs have used additional data collected to supplement original effectiveness trial findings.\n\nDBCIs require novel methods of evaluation that are quick and timely to inform the optimisation of the intervention14. The multiphase optimisation strategy (MOST) is a method for building, optimising and evaluating multicomponent behavioural interventions. It involves a series of steps identifying the set of intervention components to be examined and evaluating the effects of these components13,15. Factorial trial designs allow the simultaneous evaluation of the intervention components, which enables both the independent and interactive effects to be estimated13. Using a factorial trial to evaluate a DBCI can overcome some of the challenges associated with using the traditional randomised controlled trial, such as prolonged duration from recruitment to publication and a high-cost trial implementation16,17. The results from a factorial trial can be used to make decisions about which components to retain when optimising the intervention15.\n\nThe Drink Less smartphone app is a DBCI aimed at supporting people who drink excessively to reduce their alcohol consumption. It was developed using evidence and theory, following MOST. The app was analysed in a full factorial trial to assess the effectiveness of its five intervention modules and their effects on app usage and subsequent usability ratings18. The stopping rule for data collection, in line with the frequentist approach to analysis, was pre-specified, although data collection continued under the same conditions as the original factorial trial. Analysis of the original trial data using Bayes factors indicated that the data were insensitive to detect main effects but that combinations of the modules appeared effective1.\n\nThe aims of this study are substantive and methodological:\n\n1. To update the evidence on effectiveness of Drink Less app components singly and in combination. Specifically, what are the main and two-way interactive effects of the intervention modules on:\n\ni. Change in weekly alcohol consumption\n\nii. Change in full Alcohol Use Disorders Identification Test (AUDIT) score\n\n2 To demonstrate how Bayes Factors can be used to analyse additional outcome data collected in effectiveness trials and update beliefs about hypotheses.\n\n\nMethods\n\nA between-subject full factorial (25) trial to evaluate the effectiveness of five intervention modules in the Drink Less app. The research questions were specified prior to the trial commencing and pre-registered on ISRCTN (registration number: ISRCTN40104069) and published in an open-access protocol paper18.\n\nParticipants were included in the study if they: were aged 18 or over; lived in the UK; had an AUDIT score of 8 or above (indicative of excessive drinking19); were interested in reducing their drinking; provided an email address and had downloaded a ‘trial version’ of the app (described below).\n\nThe sample size for the original factorial trial was 672 providing 80% power (with alpha at 5%, 1:1 allocation and a two-tailed test) to detect a mean change in alcohol consumption of 5 units between the ‘enhanced’ and ‘minimal’ versions for each intervention module20, comparable with a face-to-face brief intervention21. This assumed a mean of 27 weekly units at follow-up in the control group, a mean of 22 units in the intervention group and a SD of 23 units for both (d=0.22).\n\nRecruitment was undertaken via promotion from organisations, such as Public Health England, Cancer Research UK, and listing the app in the iTunes Store according to best practices for app store optimisation.\n\nBaseline measures included the AUDIT questionnaire and a socio-demographic assessment (age, gender, ethnic group, level of education, employment status and current smoking status). The primary outcome measure was self-reported change in past week alcohol consumption (the difference between one-month follow-up and baseline). The secondary outcome measure was self-reported change in full AUDIT score.\n\nThe Drink Less app is a DBCI for people who drink excessively to help them reduce their alcohol consumption. It is freely available on the UK version of the Apple App Store for all smartphones and tablets running iOS8 or above. The content of the app did not change during the trial except for minor bug fixes.\n\nThe app is structured around goal setting: users can set their own goals based on units, cost, alcohol free days or calories with information on the UK drinking guidelines, units and alcohol-related harms. There are five intervention modules that aim to help them achieve their goal: Normative Feedback (providing normative feedback on the user’s level of drinking relative to others); Cognitive Bias Re-training (a game to retrain approach-avoidance bias for alcoholic drinks); Self-monitoring and Feedback (providing a facility for self-monitoring of drinking and receipt of feedback); Action Planning (helping users to undertake action planning to avoid drinking), and Identity Change (promoting a change in identity in relation to alcohol). In the trial version of the app, the five intervention modules existed in two versions: i) an ‘enhanced’ version containing the predicted active ingredients and ii) a ‘minimal’ version that acted as a control.\n\nA detailed description of the content, development and factorial trial evaluation of the app is reported in two separate papers1,22.\n\nData collection for the factorial trial began on 18th May 2016 and the required sample of eligible users was reached on 10th July 2016; follow-up data were collected until 28th August 2016. Trial data was collected continuously for a further four months until 19th December 2016 under the same conditions as the original factorial trial (i.e. a ‘trial version’).\n\nInformed consent to participate in the trial was obtained from all participants on first opening the app. Users who consented to participate completed the AUDIT and a socio-demographic questionnaire, indicated their reason for using the app and provided their email address for follow-up (a prize of £100 was offered in an attempt to decrease the proportion of users leaving this field blank). Users were then provided with their AUDIT score and, those who met the inclusion criteria, were randomised to one of 32 experimental conditions using an automated algorithm within the app for block randomisation.\n\nFollow-up was conducted 28 days after participants downloaded the app and the questionnaire consisted of the full AUDIT and usability measures. Follow-up was conducted in two ways: i) via email with a link to the questionnaire in an online survey tool (Qualtrics), which also sent up to four reminders, and ii) within the app. Participants included according to the original trial and stopping rule were due to complete the follow-up questionnaire up until 29th August 2016 and were contacted via email (through Qualtrics) and the app. Participants due to complete the follow-up questionnaire from 30th August onwards, were only contacted via the app.\n\nEthical approval for Drink Less from the UCL Ethics Committee under the ‘optimisation and implementation of interventions to change health-related behaviours’ project (CEHP/2013/508).\n\nAll analyses were conducted using R version 3.4.0. The analysis plan for this paper followed a similar analysis plan as for the original factorial trial (which was pre-registered on 13th February 2016; ISRCTN4010406918).\n\nParticipant characteristics were reported descriptively by intervention module. A factorial between-subjects design was used to assess the main and two-way interactive effects of the five intervention modules on the primary and secondary outcome measures. Analyses were conducted amongst responders only, those who completed the follow-up questionnaire. Bayes Factors were calculated for each analysis assessing the main and the two-way interaction effects of the five intervention modules on the outcome measures. The two-way interactions were defined as enhanced/enhanced versus minimal/minimal for each pair of intervention modules. The mean difference and standard error of the mean difference for each main and two-way interactive effect was calculated. A half normal distribution was used to specify the predicted effect. Peak at 0 (no effect) with a SD equal to the expected effect size. This is a conservative approach and represents a hypothesis that the intervention had a least some positive effect, with the effect being more likely to be smaller than larger. Bayes factors were calculated using an online calculator7.\n\nThe expected effect size for the primary calculation of Bayes factors was a reduction of 5 units per week (d=0.22), reflecting a large effect and that of the power calculation for the original factorial trial. Bayes Factors were also calculated for a medium effect (reduction of 3 units per week), and a small effect (reduction of 0.5 units per week) to permit a relative judgment for screening purposes. The expected effect size for the secondary outcome measure was calculated by translating the estimated effect size for the primary outcome measure (d=0.22) into the equivalent mean difference score of 1.45 (mean=19.1, SD=6.56 [based on original trial users, n=672]). Bayes factors will be interpreted in terms of categories of evidential strength (see Table 1)5,23.\n\nH1, alternative hypothesis; H0, null hypothesis.\n\n\nResults\n\nThe total sample size was 2586, of these 1914 (74.0%) were additional users to the original factorial trial (672, 26.0%). In total, 342 users (13.2%) completed the primary outcome measure in the follow-up questionnaire—the original users’ response rate was 26.6% and the additional users’ response rate was 8.5%. Figure 1 shows a flow chart of users throughout the study.\n\nSocio-demographic and drinking characteristics of participants are reported in Table 2. Participants’ mean age was 37.2 years, 53.4% were women, 95.8% were white, 74.3% had post-16 qualifications, 87.0% were employed, and 30.0% were current smokers. Mean weekly alcohol consumption was 39.0 units, mean AUDIT-C score was 9.3, and mean AUDIT score was 19.1, indicating harmful drinking. Participants’ characteristics by intervention module are reported in Table 2. Generally, characteristics were similar for the enhanced and minimal version of each intervention module.\n\nData given as mean (SD), unless stated.\n\nEnh, enhanced; Min, minimal.\n\nThe main effects of the intervention modules are reported in Table 3 for the change in past week’s alcohol consumption. Bayes factors showed that the data were insensitive to detect an effect for Normative Feedback for effect sizes of 5-, 3- and 0.5-unit reductions (0.47<BF<0.97). Data were insensitive to detect an effect for Cognitive Bias Re-training for effect sizes of 5-, 3- and 0.5-unit reductions (0.74<BF<1.06). Bayes factors showed that the data were insensitive to detect an effect for Self-monitoring and Feedback for effect sizes of 5-, 3- and 0.5-unit reductions (0.43<BF<0.95). Bayes factors showed that the data were insensitive to detect an effect for Action Planning for effect sizes of 5-, 3- and 0.5-unit reductions (0.83<BF<1.08). Bayes Factors for Identity Change showed support for the null hypothesis of no difference between the enhanced and minimal version of the module for a 5-unit reduction (BF=0.22), though data were insensitive to detect an effect for 3- and 0.5-unit reductions (0.34<BF<0.81). The data were insensitive to detect a two-way interactive effect between any pair of intervention modules for effect sizes of 5-, 3- or 0.5-unit reductions (0.35<BF<1.22), except for between Self-monitoring and Feedback and Identity Change for a 5-unit reduction which supported the null hypothesis (BF=0.31) (see Extended data, Supplementary Table 124).\n\nA negative number indicates a reduction over time.\n\nThe main effects of the intervention modules are reported in Table 4 for the change in AUDIT score. The data were insensitive to detect an effect on change in AUDIT score for: Normative Feedback (BF=0.60); Cognitive Bias Re-training (BF=0.98); and Action Planning (BF=0.95). The data supported evidence for the null hypothesis of no difference in AUDIT score between enhanced and minimal versions of Self-monitoring and Feedback (BF=0.15) and Identity Change (BF=0.14). The two-way interactive effects of intervention modules on change in AUDIT score (see Extended data, Supplementary Table 224) showed that the majority of data were insensitive to detect any two-way interactive effects (0.33<BF<1.99). Data supported the null hypothesis for no difference between enhanced and minimal versions between Normative Feedback and Identity Change (BF=0.29) and Self-Monitoring and Feedback and Identity Change (BF=0.18).\n\nFour intervention modules (Normative Feedback, Cognitive Bias Re-Training, Self-Monitoring and Feedback, and Action Planning) have some evidence in support of their role of reducing alcohol consumption. Therefore, an unplanned analysis was conducted to assess whether there is a larger cumulative effect of the combination of all four modules in the enhanced version compared with the minimal version. This was done for responders only (n=39; 12 “off” vs 27 “on”) and for last observation carried forward (n=324; 164 “off” vs 160 “on”) to provide potential evidence for what effect size we can expect when planning the trial. Last observation carried forward means that participants’ past week alcohol consumption at follow-up was used for all of those who responded to follow-up and the baseline measure for past week alcohol consumption was used for those who did not respond to follow-up. Whilst last observation carried forward has its limitations, it maintains the variability within the data. Table 5 reports the Bayes factors for these analyses. There was a large numerical difference between all enhanced and all minimal for the four modules amongst responders only, although the Bayes factors found that the data were insensitive to detect an effect, which may be due in part to the small sample size.\n\nPWAC, past week alcohol consumption.\n\n\nDiscussion\n\nThe calculation of Bayes factors for additional data collected beyond the original factorial trial of Drink Less has allowed us to accumulate and update existing evidence on the effectiveness of its intervention components in reducing alcohol consumption. The supplemented data remained insensitive to detect whether the Drink Less app components have large (5-unit) individual or two-way interactive effects on reducing alcohol consumption though tended towards anecdotal evidence for the null hypothesis of no effect. There was evidence of two-way interactive effects in the original factorial trial that is no longer supported by the supplemented data.\n\nThe current data also remained insensitive to detect whether the four most promising components (Normative Feedback, Cognitive Bias Re-Training, Self-Monitoring and Feedback and Action Planning) may each have effects smaller than 5 units. An unplanned analysis provided weak anecdotal evidence of a synergistic effect of the ‘enhanced’ versions of these four intervention modules together. On both past week alcohol consumption and AUDIT score, and across several alternative effect sizes, there was support for no effect of the fifth intervention module, Identity Change. These findings, alongside results from analysing user feedback and usage data on the most frequently visited screens, guided the decision to remove the Identity Change module from the next major app update whilst retaining Normative Feedback and Cognitive Bias Re-Training, and Self-Monitoring and Feedback and Action Planning.\n\nA major strength of this study is its illustration of how it is possible to evaluate data from trials of DBCIs in an on-going manner. No additional resources were required to continue data collection within the original trial of Drink Less. Analysing the supplemented dataset has allowed us to update our findings and provided more confidence in our original decisions on which components to retain or remove. The stopping rule in frequentist statistics means that additional trial data collected as part of an effectiveness trial for a DBCI would go to waste. The use of Bayes factors in this situation prevents unnecessary waste of resources and enables researchers to continually update their evidence on a DBCI rather than collect and analyse individual data sets as part of separate trials.\n\nA limitation of this study and the use of Bayes factors was that we were not able to use the intention-to-treat (ITT) approach in the analysis (as was done for the original trial), whereby those lost to follow-up (non-responders) were assumed to be drinking at baseline levels. Whilst Bayes factors can overcome a lot of the issues with the frequentist approach, they are not meaningful when assumptions are made that limit the variability in the data. Due to low overall follow-up rates (13.2%) in this larger sample, the ITT assumption that there was no change in the large majority of the sample drives the variability down, which in turn drives support for the null hypothesis. This highlights that Bayes factors were not useful in this study when using the ITT assumption, which limits the variability in the data.\n\nThe intervention modules of the Drink Less app do not have a large individual effect on reducing alcohol-related outcomes, though they may have a small effect that the current data were unable to detect. There is weak evidence for a synergistic effect of the ‘enhanced’ versions of four intervention modules together: Normative Feedback and Cognitive Bias Re-Training, and Self-Monitoring and Feedback and Action Planning. This study has updated the existing evidence on the effectiveness of intervention modules in the Drink Less app. In the event of uncertain results following a primary analysis, Bayes factors can be used to ‘top-up’ results from DBCI trials with any additional data collected, therefore supporting efficient, incremental model building to inform decision-making.\n\n\nData availability\n\nA dataset containing the extended trial outcomes is available on OSF. DOI: https://doi.org/10.17605/OSF.IO/KQM8B24.\n\nExtended data are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/KQM8B24.\n\nSupplementary Table 1. Two-way interactive effects of intervention modules on change in past week’s alcohol consumption.\n\nSupplementary Table 2. Two-way interactive effects of intervention modules on change in AUDIT score.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nJB and RW are funded by Cancer Research UK (CRUK; C1417/A22962). CG and SM are funded by CRUK and the National Institute for Health Research (NIHR)’s School for Public Health Research (SPHR). Drink Less was funded by NIHR SPHR, the UK Centre for Tobacco and Alcohol Studies (UKCTAS), the Society for the Study of Addiction (SSA), and CRUK. The views expressed are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.\n\nThe research team is part of the UKCTAS, a UKCRC Public Health Research Centre of Excellence. Funding from the Medical Research Council, British Heart Foundation, Cancer Research UK, Economic and Social Research Council and the National Institute for Health Research under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged.\n\nThe funders played no role in the design, conduct or analysis of the study, nor in the interpretation or reporting of study findings.\n\n\nReferences\n\nCrane D, Garnett C, Michie S, et al.: A smartphone app to reduce excessive alcohol consumption: Identifying the effectiveness of intervention components in a factorial randomised control trial. Sci Rep. 2018; 8(1): 4384. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRouder JN: Optional stopping: No problem for Bayesians. Psychon Bull Rev. 2014; 21(2): 301–8. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarnett C, Crane D, Michie S, et al.: Evaluating the effectiveness of a smartphone app to reduce excessive alcohol consumption: protocol for a factorial randomised control trial. BMC Public Health. 2016; 16(1): 536. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReinert DF, Allen JP: The alcohol use disorders identification test: an update of research findings. Alcohol Clin Exp Res. 2007; 31(2): 185–99. PubMed Abstract \| Publisher Full Text\n\nKunz FM Jr, French MT, Bazargan-Hejazi S: Cost-effectiveness analysis of a brief intervention delivered to problem drinkers presenting at an inner-city hospital emergency department. J Stud Alcohol. 2004; 65(3): 363–70. PubMed Abstract \| Publisher Full Text\n\nKaner E, Beyer F, Dickinson HO, et al.: Effectiveness of brief alcohol interventions in primary care populations. Cochrane Database Syst Rev. 2007; (2): CD004148. PubMed Abstract \| Publisher Full Text\n\nGarnett C, Crane D, West R, et al.: The development of Drink Less: an alcohol reduction smartphone app for excessive drinkers. Transl Behav Med. 2018. PubMed Abstract \| Publisher Full Text\n\nAndraszewicz S, Scheibehenne B, Rieskamp J, et al.: An Introduction to Bayesian Hypothesis Testing for Management Research. J Manage. 2015; 41(2): 521–43. Publisher Full Text\n\nGarnett C, Michie S, West R: Updating evidence of effectiveness of Drink Less using Bayes factors to analyse additional outcome data. 2019. http://www.doi.org/10.17605/OSF.IO/KQM8B" }	[ { "id": "43750", "date": "11 Feb 2019", "name": "Daniel Leightley", "expertise": [ "Reviewer Expertise Mobile health with a focus on alcohol misuse" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the effectiveness of DrinkLess, a mobile alcohol intervention, using Bayes factor to further compliment previously published findings. I have one major comment and several minor comments.\nMajor Comment:\nThe work extends our understanding of DrinkLess and its effectiveness in managing alcohol misuse; however, it would be helpful to make a clear statement on how the Bayes analysis has improved the value proposition of DrinkLess.\nMinor Comments:\nAbstract: “Amongst responders only” – is that the sample who took part in the follow-up questionnaire?; Abstract: “Unplanned comparison” appears to convey a negative connotation the authors could alter to “additional analyses”; Abstract: “four most promising” could be misleading as you only had five components but we also have to be mindful that the data was insensitive; Abstract: “reminded insensitive but tended” are you able to provide any BF for this statement?; Introduction: It would be helpful to provide some discussion (specific examples) on how Bayes have been used in other domains to provide more insight by means of additional data; Methods – Participants: “were interested in reducing their drinking” how was this measured? Were participants research aware, or were they targeted because they had previously stated an interest in reducing alcohol? Or could it be by downloading DrinkLess they were assumed to be interested in reducing their alcohol consumption?; Methods – Participants: Was a geolocation restriction placed on participants? How can you be sure that users were from the UK?; Methods – Intervention: What were the minor bug fixes, is a summary able to be provided as a supplement?; Results: Results present AUDIT-C score, however this is not discussed previously. Results: It would be helpful to have Table 2 represented as supplementary material for those who took part in follow-up; Results: It would be of interest to discuss further the difference in AUDIT and AUDIT-C score and the role the final two questions (risk taking etc) play; Discussion: “no additional resources were required” – is this the case, was the app provisioned for longer than anticipated?; Discussion: “our decision on which components to retain or remove” – a bit more discussion round this aspect would be helpful to the reader; Discussion: A 13.2% follow-up rate appears to be very low, do the authors have any reasons for this?;\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": "4718", "date": "15 Jul 2019", "name": "Claire Garnett", "role": "Author Response", "response": "The work extends our understanding of DrinkLess and its effectiveness in managing alcohol misuse; however, it would be helpful to make a clear statement on how the Bayes analysis has improved the value proposition of DrinkLess.This is a very good point made by both reviewers that we do not sufficiently discuss the value proposition of Drink Less in our discussion section. We have added a paragraph on this:“Whilst this study did not find evidence of a large individual effect of any of the intervention modules, there remains some evidence to suggest that an optimised version of the app (with the removal of the ‘Identity Change’ module) may yet prove effective. As with the original factorial trial, there are concerns that the minimal versions were too active in an attempt to promote engagement amongst all participants. Even participants who were randomised to receive the minimal versions of every intervention module were able to set goals and track their drinks, which is associated with reduced consumption [1]. Most alcohol reduction apps include few techniques to change behaviour [2] suggesting that even the minimal version of Drink Less was more active than most existing alcohol reduction apps. Therefore, effectiveness estimates derived from this approach are likely to be conservative. Furthermore, Drink Less users have excellent levels of engagement with the app [3], which is necessary (but not sufficient) for an intervention to be effective. Additionally, a content analysis of user feedback (available as a short report here: https://osf.io/d3w8r/) found that of the ‘Information giving’ category, the majority provided positive feedback on the app as a whole. A sample of the user feedback is available to view on the Drink Less website [4]. Drink Less is also one of the leading alcohol reduction apps in the UK with over 50,000 unique users to date with an average 4.1-star rating (as of June 2019).”Abstract: “Amongst responders only” – is that the sample who took part in the follow-up questionnaire?;Yes, this has been clarified in the abstract with “…amongst responders only (those who completed the questionnaire).”Abstract: “Unplanned comparison” appears to convey a negative connotation the authors could alter to “additional analyses”;We have reworded this to “additional exploratory analysis” throughout the manuscript.Abstract: “four most promising” could be misleading as you only had five components but we also have to be mindful that the data was insensitive;We have re-worded the abstract to be cautious with our conclusions:“In an additional exploratory analysis, participants receiving four of the components averaged a numerically greater reduction in consumption than those not receiving any (21.6 versus 12.1 units), but the data were insensitive (BF=1.42).Data from extended recruitment in a factorial experiment evaluating components of the Drink Less app remained insensitive but tended towards individual and pairs of components not having a large effect. In an exploratory analysis, there was weak anecdotal evidence for a synergistic effect of four components. In the event of uncertain results, calculating BFs can be used to update the strength of evidence of a dataset supplemented with extended recruitment.”Abstract: “reminded insensitive but tended” are you able to provide any BF for this statement?;The relevant BFs for this conclusion are included in the results section of the abstract and we have added the BF range for the two-way interactive effects: “Data were mainly insensitive but tended to support there being no large main effects of the enhanced version of individual components on consumption (0.22Introduction: It would be helpful to provide some discussion (specific examples) on how Bayes have been used in other domains to provide more insight by means of additional data;We have added further details into the introduction about previous uses of Bayes factors and Bayesian analyses:“To the authors’ knowledge, no DBCIs have used additional data collected to supplement original effectiveness trial findings and no trials have used Bayes Factors to provide further insight based on additional data. However, Bayes Factors have been used in trials for superiority, non-inferiority and equivalence designs to allow for explicit quantification of evidence in favour of the null hypothesis [1]. Bayesian analyses, more generally, are often used in clinical trials for dose finding, efficacy monitoring, toxicity monitoring, and for diagnosis/decision making [2]. For example, Bayesian analyses were used to simultaneously monitor toxicity and efficacy in a parallel phase I/II clinical trial design for combination therapies [3].”Methods – Participants: “were interested in reducing their drinking” how was this measured? Were participants research aware, or were they targeted because they had previously stated an interest in reducing alcohol? Or could it be by downloading DrinkLess they were assumed to be interested in reducing their alcohol consumption?;Methods – Participants: Was a geolocation restriction placed on participants? How can you be sure that users were from the UK?;We have clarified details on the participants in the methods section:“Participants were included in the study if they: were aged 18 or over; lived in the UK (only available on UK Apple app store and users had to select ‘UK’ for ‘Country?’); had an AUDIT score of 8 or above (indicative of excessive drinking); were interested in reducing their drinking (indicated by the question ‘why are you using this app?’ with users choosing ‘interested in drinking less’ over ‘just browsing’); provided an email address and had downloaded a ‘trial version’ of the app (described below).”Methods – Intervention: What were the minor bug fixes, is a summary able to be provided as a supplement?;As the bug fixes are minor, we have included a brief explanation in the manuscript: “The content of the app did not change during the trial except for minor bug fixes (to ensure compatibility with iOS 10).”Results: Results present AUDIT-C score, however this is not discussed previously.We have clarified this in the measures section:“The primary outcome measure was self-reported change in past week alcohol consumption (the difference between one-month follow-up and baseline). Past week alcohol consumption was derived from the frequency (Q1) and quantity (Q2) questions of the AUDIT-Consumption (AUDIT-C) questionnaire.”Results: It would be helpful to have Table 2 represented as supplementary material for those who took part in follow-up;We have added a supplementary table of the participant characteristics for those who responded to follow-up.Results: It would be of interest to discuss further the difference in AUDIT and AUDIT-C score and the role the final two questions (risk taking etc) play;We have discussed the differences between the AUDIT-C and AUDIT scores in the methods section:“The secondary outcome measure was self-reported change in full AUDIT score; in addition to the three questions on consumption in the AUDIT-C, the full AUDIT includes questions assessing harmful alcohol use (e.g. alcohol-related injuries) and symptoms of dependence.”Discussion: “no additional resources were required” – is this the case, was the app provisioned for longer than anticipated?;The app was always intended to be available for the long-term (i.e. not removing it after completion of the trial). We have added more explanation to this section of the discussion:“No additional resources were required to continue data collection within the original trial of Drink Less as the app remained freely available on the UK Apple app store and the notification to complete the follow-up questionnaire had already been programmed.”Discussion: “our decision on which components to retain or remove” – a bit more discussion round this aspect would be helpful to the reader;We have elaborated on this in the discussion:“Analysing the supplemented dataset has allowed us to update our findings and provided more confidence in our original decisions on which components to retain or remove as part of the process of optimising the intervention 15 to improve its effectiveness and usability. We are also much clearer that any definitive trial must be powered to detect small effects and designed to inform a pragmatic decision about whether to invest resources in recommending the app. The optimisation of the Drink Less intervention was based on the findings from this study as well as on user feedback and findings from a meta-analysis of the intervention components in digital alcohol interventions associated with effectiveness [9]. The findings from this study informed the removal of the ‘Identity Change’ module and retention of the remaining four modules.”Discussion: A 13.2% follow-up rate appears to be very low, do the authors have any reasons for this?;We have included a discussion of this in the limitations:“We acknowledge that the follow-up rate is very low and this is likely to be due to the lack of financial incentive for completing the follow-up survey, which are known to increase response rates in randomised trials (Brueton et al., 2014). Furthermore, the follow-up rate in the extended dataset was lower than for the original trial dataset; this is likely because participants were only contacted via the app for the extended dataset whilst the participants in the original dataset were also contacted via email.”" } ] }, { "id": "44925", "date": "13 Mar 2019", "name": "Nick Heather", "expertise": [ "Reviewer Expertise I have long experience in the area of research on brief interventions for hazardous and harmful alcohol consumption. However", "although I have authored a publication using the Bayesian approach to hypothesis testing", "I am by no means an expert on the use of Bayesian 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\nThis paper reports the results of a factorial experiment evaluating the Drink Less Smart phone app and its behaviour change components, with the benefit of an extended data-set. The aims of the study were both substantive and methodological. In relation to the latter, the paper provides a very useful example of the advantages of Bayesian hypothesis testing and in particular its legitimate provision for extending data collection until a firm conclusion has been reached. The paper also provides a good example, with earlier papers, of the MOST method for developing and evaluating behaviour change interventions.\nRegarding the substantive aim, I have two major and several minor comments.\nMAJOR:\nAnalysis of the full data-set after extension confirmed the mainly inconclusive results of tests of the 5 individual components of the app in the original evaluation study and failed to confirm the previously reported evidence in favour of two interaction effects between components. In view of the authors' thorough and painstaking development of the app over the years, their rigorous evaluations of the components of the app and their stated intention to optimise the app for a definitive test in an RCT with extended follow-up, these results must be regarded as very disappointing. Yet this is not commented on and no possible explanation is offered why components whose inclusion was supported strongly by theory and previous research failed to show effects on drinking behaviour. Is it possible, as the authors have previously suggested, that the 'minimal', control interventions were too active to allow an effect to emerge? What other explanations are there for these disappointing results? Overall, where do the authors go from here in the attempt to bring this very promising intervention technology to practical use? In the Abstract the authors state: 'There was weak evidence for a synergistic effect of four components'. I feel even this is too strong. In the text on p.9 we have: 'An unplanned analysis provided weak anecdotal evidence of a synergistic effect of the ‘enhanced’ versions of these four intervention modules together'. Something along these lines, with the inclusion of 'unplanned' and 'anecdotal' would be more appropriate for the Abstract.\n\nAnother concern here is that the putative effect in question derives from a post hoc hypothesis based on unplanned comparisons arrived at only after the extension of data collection. In a frequentist approach to hypothesis testing, this would not of course be permissible but, even in the Bayesian approach, there must surely be some constraints on the legitimacy of testing post hoc hypotheses derived from exploratory analyses (rather than using such analyses to generate hypotheses not tested in the present data). The authors should comments on this issue and, if necessary, seek expert advice.\nMINOR:\nWhy was the follow-up rate so much lower in the extended data-set that in the original data (8.5% versus 26.6%)? Can the authors attempt to explain this difference? p.8: '... to provide potential evidence for what effect size we can expect when planning the trial.' This is presumably the definitive trial with extended follow-up but this is not clear. What about the data on secondary outcomes? These are not reported here but it is not stated that they will not be considered in this paper and the reader is not told where they will be found. The primary outcome measure of self-reported change in past week alcohol consumption was presumably based on the AUDIT-C questionnaire, as suggested by Table 2, but this is not made clear in the text.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "4719", "date": "15 Jul 2019", "name": "Claire Garnett", "role": "Author Response", "response": "Analysis of the full data-set after extension confirmed the mainly inconclusive results of tests of the 5 individual components of the app in the original evaluation study and failed to confirm the previously reported evidence in favour of two interaction effects between components. In view of the authors' thorough and painstaking development of the app over the years, their rigorous evaluations of the components of the app and their stated intention to optimise the app for a definitive test in an RCT with extended follow-up, these results must be regarded as very disappointing. Yet this is not commented on and no possible explanation is offered why components whose inclusion was supported strongly by theory and previous research failed to show effects on drinking behaviour. Is it possible, as the authors have previously suggested, that the 'minimal', control interventions were too active to allow an effect to emerge? What other explanations are there for these disappointing results? Overall, where do the authors go from here in the attempt to bring this very promising intervention technology to practical use?This is a very good point made by both reviewers that we do not sufficiently discuss the value proposition of Drink Less in our discussion section. We have added a paragraph on this:“Whilst this study did not find evidence of a large individual effect of any of the intervention modules, there remains some evidence to suggest that an optimised version of the app (with the removal of the ‘Identity Change’ module) may yet prove effective. As with the original factorial trial, there are concerns that the minimal versions were too active in an attempt to promote engagement amongst all participants. Even participants who were randomised to receive the minimal versions of every intervention module were able to set goals and track their drinks, which is associated with reduced consumption [1]. Most alcohol reduction apps include few techniques to change behaviour [2] suggesting that even the minimal version of Drink Less was more active than most existing alcohol reduction apps. Therefore, effectiveness estimates derived from this approach are likely to be conservative. Furthermore, Drink Less users have excellent levels of engagement with the app [3], which is necessary (but not sufficient) for an intervention to be effective. Additionally, a content analysis of user feedback (available as a short report here: https://osf.io/d3w8r/) found that of the ‘Information giving’ category, the majority provided positive feedback on the app as a whole. A sample of the user feedback is available to view on the Drink Less website [4]. Drink Less is also one of the leading alcohol reduction apps in the UK with over 50,000 unique users to date with an average 4.1-star rating (as of June 2019).”In the Abstract the authors state: 'There was weak evidence for a synergistic effect of four components'. I feel even this is too strong. In the text on p.9 we have: 'An unplanned analysis provided weak anecdotal evidence of a synergistic effect of the ‘enhanced’ versions of these four intervention modules together'. Something along these lines, with the inclusion of 'unplanned' and 'anecdotal' would be more appropriate for the Abstract.We have re-worded the abstract to be cautious with our conclusions:“In an additional exploratory analysis, participants receiving four of the components averaged a numerically greater reduction in consumption than those not receiving any (21.6 versus 12.1 units), but the data were insensitive (BF=1.42).Data from extended recruitment in a factorial experiment evaluating components of the Drink Less app remained insensitive but tended towards individual and pairs of components not having a large effect. In an exploratory analysis, there was weak anecdotal evidence for a synergistic effect of four components. In the event of uncertain results, calculating BFs can be used to update the strength of evidence of a dataset supplemented with extended recruitment.”Another concern here is that the putative effect in question derives from a post hoc hypothesis based on unplanned comparisons arrived at only after the extension of data collection. In a frequentist approach to hypothesis testing, this would not of course be permissible but, even in the Bayesian approach, there must surely be some constraints on the legitimacy of testing post hoc hypotheses derived from exploratory analyses (rather than using such analyses to generate hypotheses not tested in the present data). The authors should comments on this issue and, if necessary, seek expert advice.These analyses were exploratory rather than testing a post-hoc hypothesis and the Bayesian approach does not have the same limitations as the frequentist approach in conducting exploratory analyses. Also, the conclusions match the strength of the evidence for this exploratory analysis (weak and anecdotal). We plan to pre-register any hypotheses and analysis plans in future studies. Furthermore, a key part of this study was for it to be informative in making decisions on which components to retain in an optimised version of the app and in terms of conducting a power analysis for a definitive trial.Why was the follow-up rate so much lower in the extended data-set that in the original data (8.5% versus 26.6%)? Can the authors attempt to explain this difference?We have included a discussion of this in the limitations:“We acknowledge that the follow-up rate is very low and this is likely to be due to the lack of financial incentive for completing the follow-up survey, which are known to increase response rates in randomised trials (Brueton et al., 2014). Furthermore, the follow-up rate in the extended dataset was lower than for the original trial dataset; this is likely because participants were only contacted via the app for the extended dataset whilst the participants in the original dataset were also contacted via email.”p.8: '... to provide potential evidence for what effect size we can expect when planning the trial.' This is presumably the definitive trial with extended follow-up but this is not clear.We have clarified this:“…to provide potential evidence for what effect size we can expect when planning a definitive trial with longer-term follow-up.”What about the data on secondary outcomes? These are not reported here but it is not stated that they will not be considered in this paper and the reader is not told where they will be found.The data on the other secondary outcomes (usage data and usability ratings) were not reported in this paper though we have added information on where they will be found:“Other secondary outcome measures included in the original, full factorial trial were usage data and usability ratings though were not considered in this paper. Details of these measures are described elsewhere 1, and the data and Bayes Factors calculated are reported on the Open Science Framework (https://osf.io/kqm8b/).”The primary outcome measure of self-reported change in past week alcohol consumption was presumably based on the AUDIT-C questionnaire, as suggested by Table 2, but this is not made clear in the text.We have clarified this in the measures section:“The primary outcome measure was self-reported change in past week alcohol consumption (the difference between one-month follow-up and baseline). Past week alcohol consumption was derived from the frequency (Q1) and quantity (Q2) questions of the AUDIT-Consumption (AUDIT-C) questionnaire.”" } ] } ]	1	https://f1000research.com/articles/8-114
https://f1000research.com/articles/8-1073/v1	15 Jul 19	{ "type": "Case Report", "title": "Case Report: Morphine withdrawal induced convulsions in an adult male patient", "authors": [ "Mahmoud M. Ali", "Abdelrahman Hamad", "Eman Nawash Alhamoud", "Abdelrahman Hamad", "Eman Nawash Alhamoud" ], "abstract": "This case report describes a possible unknown complication of morphine withdrawal in a patient with persistent back pain, treated with intrathecal morphine pump infusion. The patient presented with left lower extremity edema. After excluding deep vein thrombosis by Doppler ultrasound and worsening of the swelling despite oral antibiotics, peripheral edema caused by intrathecal morphine was suspected. Twelve hours following the termination of his intrathecal morphine pump and initiation of inequivalent doses of oral morphine and tramadol, he developed convulsions. After metabolic and structural causes of convulsion were ruled out by blood tests and head imaging, equivalent doses of morphine were given. Then the patient regained full consciousness, and no additional seizures occurred. After that, opioid withdrawal emerged as the most likely explanation. Seizure is a life-threating condition; therefore, an awareness of this case is important and further studies are warranted to explore the potential association of opioid withdrawal and seizure.", "keywords": [ "Morphine", "Seizure", "Withdrawal", "Intrathecal morphine", "Opioid", "Convulsion" ], "content": "Introduction\n\nMorphine withdrawal is a common medical problem. Patients with morphine withdrawal can present with a variety of symptoms including runny nose, watery eyes, fever, vomiting, nausea, headaches, sweating, chills, muscle aches, diarrhea, high blood pressure, agitation, anxiety, irritability, depression, disorientation, insomnia1. This case report describes a seizure as a clinical complication during an adult male patient’s withdrawal from morphine. The link between opioid withdrawal and seizures is not well studied in adult humans. To the best of our knowledge, only two case series of seven patients and three patients have been reported tying opioid withdrawal to seizures2,3.\n\n\nCase presentation\n\nA 44-year-old Qatari man known to have persistent back pain admitted to our facility in 2017. He presented with left lower extremity edema that started approximately three to four weeks prior to admission. It was affecting his daily activities like showering and driving. The edema began in his foot and then gradually progressed to his abdomen. A physical examination found soft pitting edema in the left lower limb up to the sacrum posteriorly and to the umbilicus anteriorly. His lower limb showed some redness with no hotness, tenderness, or signs of chronic venous insufficiency. His past surgical history demonstrated multiple back surgeries, as follows; in 1986, he underwent surgical correction and fusion of lumbar scoliosis anteriorly and posteriorly. Additionally, in 1986, he had triple arthrodesis of his right foot. In 1992, he underwent lengthening of his atrophic flail right leg. In 1993, the Harrington rod from the dorsal and lumbar spine was removed. In 2004, he had anterior lumbar interbody fusion with cages at the levels of T10-T11 and T11-T12. In 2008, he underwent revision surgery to extend the anterior instrumentation from T2 to T12. In March of 2014, the patient had intrathecal morphine pump inserted with a morphine infusion rate of 5.75 mg/day.\n\nUpon admission, a Doppler ultrasound scan of his left lower limb revealed no evidence of deep vein thrombosis (Figure 1). The patient was started empirically on amoxicillin-clavulanic acid (875 mg orally every 12 hours for five days), suspecting community-acquired cellulitis as one of the common causes of unilateral lower limb edema, but his edema did not improve. On the fifth day of admission, the patient started to develop new edema on the right leg. A pelvic and abdominal ultrasound scan showed no obvious mass (Figure 2). We then suspected that his intrathecal morphine infusion may be the cause of his peripheral edema, as other common causes were excluded, so the morphine pump was halted, and the pain management team initiated the patient on oral morphine (30 mg) twice daily and tramadol (50 mg) every six hours.\n\nA and B) left common femoral vein; C, D and E) left superficial femoral vein; F) left popliteal.\n\nA) urinary bladder; B) urinary bladder postvoid; C) mid abdomen; D) gallbladder; E) right kidney; F) left kidney; G) spleen.\n\nAfter twelve hours from pump termination, the patient started to convulse. He had three episodes of convulsion over two hours in the form of generalized tonic-clonic convulsion with rolled-up eyes; each episode was preceded by progressive muscle twitches. They were associated with continuous high blood pressure, ranging from 180/100 mmHg to 210/110 mmHg, and profuse sweating. All of the seizure episodes were aborted within a few seconds following the administration of 5mg intravenous diazepam, which was administered one to two minutes after the seizure started. Four hours later, another three seizure episodes occurred. The first was aborted by 5mg intravenous diazepam and the other two episodes required 10mg of intravenous diazepam.\n\nA computed tomography review of the patient’s head was grossly normal and revealed no acute intracranial event (Figure 3). A complete metabolic panel was done and revealed no acute metabolic process or hypoglycemia. The patient’s morphine regimen changed to 5 mg administered intravenously every four hours with oral tramadol (50 mg) every six hours. In the evening, the patient regained full consciousness and no additional seizures occurred.\n\nA–E) Computed tomography of the patient’s head.\n\nUpon patient request, the intrathecal morphine pump was restarted. One day after, the patient’s swelling in left lower limb started to increase so the intrathecal morphine pump was stopped, and the patient was started on patient-controlled analgesia fentanyl (50 mcg/hour) and oral methadone (10 mg every six hours). His left- and right-side edema disappeared gradually over seven days and after he regained his baseline functional capacity, he was discharged.\n\n\nDiscussion\n\nCentral nervous system irritability is a known opioid withdrawal sign in neonates4 and is accompanied by seizures in 2% to 11% of cases5,6. While a high degree of cerebral activity and seizure has been reported in rodent model opioid withdrawal studies7, the link between opioid withdrawal and seizures is not well studied in adult humans. To the best of our knowledge, only two case series of seven patients and three patients have been reported tying opioid withdrawal to seizures2,3.\n\nOur patient was not known to be an opioid addict from their history and their opioid risk tool score of one8, so the concurrent use of another known seizure-inducing substance was unlikely. He was receiving an intrathecal dose of morphine which changed to an unequal oral dose of morphine, in addition to tramadol. Seizures are not mentioned in the literature as a known complication of morphine withdrawal, and the patient’s complication may have been caused by severe pain accompanied by inadequate doses of analgesics.\n\n\nConclusions\n\nThis case illustrates a possible connection between opioid withdrawal and seizure in an adult male patient. Seizure is a life-threating condition; therefore, an awareness of this case is important and further studies are warranted to explore the potential association of opioid withdrawal and seizure.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSadock BJ, Sadock VA, Ruiz P: Kaplan and Sadock's Synopsis of Psychiatry. 11th ed. New Delhi: Wolter Kluwer; 2015. Reference Source\n\nParkar S, Seethalakshmi R, Adarkar S, et al.: Is this ‘complicated’ opioid withdrawal? Indian J Psychiatry. 2006; 48(2): 121–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJain S, Singhai K, Swami M: Seizure as a primary presentation in opioid withdrawal. Psychiatry Clin Neurosci. 2018; 72(10): 802–803. PubMed Abstract \| Publisher Full Text\n\nHerzlinger RA, Kandall SR, Vaughan HG Jr: Neonatal seizures associated with narcotic withdrawal. J Pediatr. 1977; 91(4): 638–641. PubMed Abstract \| Publisher Full Text\n\nZelson C, Rubio E, Wasserman E: Neonatal narcotic addiction: 10 year observation. Pediatrics. 1971; 48(2): 178–189. PubMed Abstract\n\nKandall SR, Gartner LM: Late presentation of drug withdrawal symptoms in newborns. Am J Dis Child. 1974; 127(1): 58–61. PubMed Abstract \| Publisher Full Text\n\nPinsky C, Dua AK, LaBella FS: Peptidase inhibitors reduce opiate narcotic withdrawal signs, including seizure activity, in the rat. Brain Res. 1982; 243(2): 301–7. PubMed Abstract \| Publisher Full Text\n\nWebster LR, Webster RM: Predicting aberrant behaviors in Opioid-treated patients: preliminary validation of the Opioid risk tool. Pain Med. 2005; 6(6): 432–442. PubMed Abstract \| Publisher Full Text" }	[ { "id": "52978", "date": "09 Sep 2019", "name": "Johnson Pradeep Ruben", "expertise": [ "Reviewer Expertise Addiction Psychiatry", "Child Psychiatry", "Resilience in wifes of alcoholism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting and very useful. There are few suggestions.\nThere needs to be more details about the use of opioids in the past surgeries, whether the patient was dependent to Opioids in the past?\n\nDid the patient have status epilepticus?\n\nMore information about what blood investigations were done and details about the same.\n\nDiscussion is very superficial and the pathophysiology of seizures in a opioid withdrawal needs to be discussed.\nKindly answer the above questions.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "4913", "date": "10 Sep 2019", "name": "Mahmoud Ali", "role": "Author Response", "response": "Thank you for your informative review The patient used the opioid “morphine and fentanyl” during the post-operative periods only. The doses during those times are not well accurate. Our patient was not known to be an opioid addict from their history and their opioid risk tool score of one The patient did not have status epilepticus, as he was regaining his full conscious in between the attacks. Blood investigations which sent were; Complete blood count. A complete metabolic panel which includes “renal function tests, Liver function tests, calcium, phosphorus, magnesium, and albumen.” Random blood sugar. Sepsis workup which includes “blood cultures, CRP, and procalcitonin.” Seizures in an opioid withdrawal in adults are not well known, and there is no much data about its pathophysiology." } ] }, { "id": "66498", "date": "13 Jul 2020", "name": "Carlos Arias Morales", "expertise": [ "Reviewer Expertise Internal medicine", "clinical medicine research", "neuroscience 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\nAli and collaborators describe a case of a possible association between opioid withdrawal and seizure-like activity. As stated by the authors, there are only a few cases reported in the literature that associate opioid use and this type of complication.\n\nThe case history and progression has been described in detail. However, there are some points that need to be addressed. Surgical history could be summarized in a way that readers have the idea of an extensive orthopedic surgery history without providing specific details about those procedures, such as the year when they were performed.\n\nIt is stated by the authors that the pain management team initiated the oral opioid treatment after the discontinuation of intrathecal pump. It would be helpful to know if the team made the switch based on opioid conversion tables to provide the patient with equianalgesic doses. Additionally, it will be helpful to know if a urine drug screen was performed upon admission to rule out illegal substance use that could potentially cause seizures.\n\nThe discussion section needs to be extended. The authors state there is a lack of literature regarding the association between opioid withdrawal and seizure-like activity. However, there have been some articles proposing a potential mechanism for which seizures may occur in those cases (Khanra et al., 20151).\n\nAlso, the authors stated that in their case the seizure may have been caused by severe pain and inadequate doses of analgesics. However, when making conclusions they state that the case illustrates a possible connection between opioid withdrawal and seizure, which creates a contradiction. It is advised that the authors review the cited article on this report regarding potential mechanism of seizure-like activity in chronic opioid use. Lastly, the manuscript needs to be revised for grammar and semantics.\n\nKindly revise the above observations.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1073
https://f1000research.com/articles/7-1936/v1	16 Dec 18	{ "type": "Research Article", "title": "Proximate and antioxidant activities of bio-preserved ogi flour with garlic and ginger", "authors": [ "Abiola F. Olaniran", "Sumbo H. Abiose", "Sumbo H. Abiose" ], "abstract": "Background: Ogi from locally available cereals remains a relatively affordable complementary food in West Africa, but has a tendency to spoil due it high moisture content. This study explored effects of garlic and ginger as biopreservatives in ogi flour. Methods: Ogi flour was prepared from sorghum and quality protein maize grains with different concentrations of garlic and ginger powder (2 and 4% w/w) by fermentation technique. These samples were stored for 16 weeks during which the total titratable acidity, pH, proximate composition, mineral content and total antioxidant activities were determined. Results: The proximate compositions of bio-preserved ogi samples were relatively stable throughout storage. The addition of garlic and ginger slightly increased the ash (0.04%), crude protein and mineral contents (mg/ 100g) of the samples. Magnesium (10.85-13.13 and 5.17-9.72); zinc (1.37-1.78 and 7.01-8.50), manganese (1.30-1.71 and 0.45-0.86) and iron (1.53-1.77 and 0.68-2.77) contents increased on addition (of garlic and ginger) to maize ogi and sorghum ogi flours respectively. The free radical scavenging activity; total phenolic and flavonoid contents increased correspondingly with the antioxidants activity. Conclusion: Although not well known to ogi consumer, the bio-preserved ogi flours showed better nutritional values and have potential as a health food.", "keywords": [ "Garlic", "Ginger", "Antioxidants", "Quality", "Ogi flour", "Biopreservation" ], "content": "Introduction\n\nCereal grains have become the most important plant group in terms of the human diet1. Sorghum is crucial in developing countries food security because it’s one of the most important staple foods for millions of poor rural people in the semiarid tropics of Asia and Africa2. Nigeria is the largest sorghum producer in 2016 in West and Central Africa region which accounts for approximately 23% of its production in Africa with forecast indicating it will become the largest sorghum grain producer in the world by 20203,4. Sorghum is rich in fats, protein, fiber, and minerals such as potassium, phosphorus, iron and calcium2. Sorghum is gluten-free, and therefore a suitable alternative grain for people with gluten intolerance5. Maize and sorghum are important cereal crops in Africa and Asia, and are consumed in various ways (porridges, snacks etc.)6. They are the main staples of the majority of the Nigerian populace. Maize contains natural bioactive chemical compounds such as carotenoids and phenolic compounds7. Quality protein maize (QPM) is nutritionally superior over the normal maize due to balanced amounts of essential amino acids, with a high content of lysine and tryptophan, and low content of leucine and isoleucine8. Quality protein maize contains the optimal amount of amino acids in protein intake when compared with the amino acid composition to egg protein9. Replacement of normal maize with quality protein maize (QPM) will impart better nutritional value to the consumers, due to its higher tryptophan (55%) and lysine (30%) content compared to normal maize. This will also contribute to food and nutrition security of the poor communities, and improve linear growth in weaning children by 19.3% where maize is consumed as staple food1. Ogi is a thin gruel commonly used as a breakfast cereal and for infant weaning food in West Africa because it is readily available, cheap, and can be produced at household level. It can be prepared by fermentation of maize, sorghum and millet10,11. Ogi in paste form has the tendency to spoil because of its high moisture content12. Garlic and ginger can be used biopreservative due to their antibacterial and antifungal properties to extend the shelf life of food13. Garlic is found almost all over the world, and is an important herb which is now an integral part of human diet and has also been linked to health benefit such as anticancer, antioxidant, therapeutic effect, stimulation of digestion, and absorption of food14,15. Ginger is also widely used around the world in food as a spice. Both are generally regarded as safe (GRAS) for consumption in food16. There is need to preserve ogi with naturally available spices, such as garlic and ginger, that are widely used and available. The study focused on the effect of garlic and ginger on the nutritionals quality of ogi flour based on the proximate and mineral composition, in addition to its antioxidant activities during storage, with the aim of preserving and enhancing the nutritional level.\n\n\nMethods\n\nChemicals and materials used in this study were of analytical grade namely; 1,1-diphenyl-2-picrylhydrazyl radical (Sigma Aldrich, Germany, D9132), Gallic acid (Sigma Aldrich, Germany, G7384), Quercetin (Sigma Aldrich, Germany), Folin Ciocalteau phenol reagent (Sigma Aldrich, Germany, F9252), Aluminum chloride (BDH, England,101084), potassium acetate (Sigma Aldrich, Germany, 791733-500G), sulfuric acid (38308-1EA), Ethanol (BDH, England, BDH1156-4LP), phenolphthalein (Sigma Aldrich, 74760-100ML), sodium hydroxide (Sigma Aldrich, 38227 1EA), hexane (Sigma Aldrich, Germany, C100307-2.5L) Boric acid (Sigma Aldrich, Germany,38750-1EA), Hydrochloric (Sigma Aldrich, Germany, 382801EA), Whatman No.1 filter paper (28413923) supplied by Finlab Nigeria Limited and Equilab Business solution Limited Nigeria.\n\nTwo hundred and fifty (250) grams of garlic bulbs and ginger rhizomes that were freshly harvested were washed, drained, peeled, diced into cubes, and dried at 65 °C for 12 h using hot air oven (Gallenkamp, UK). They were then ground using a grinder (Marlex Appliances PVT, Mumbai, India). Powdered garlic and ginger passed through a sieve (60 µm) (BS mesh sieves, Dual manufacturing Co. Chicago, USA) for removal of residues13.\n\nQuality protein maize (ART/98/SW06/OB/W) was obtained from the Institute of Agricultural Research and Training (I.A.R.T.), Ibadan, Nigeria, while sorghum was procured from a local market in Ile – Ife, Osun State, Nigeria. 15 kilogram of grains were examined, winnowed, and steeped separately for 3 days. Milling into smooth paste after draining of the grains was done with an attrition mill (No 1 Premier mill, England). Powdered garlic and ginger were added for co-fermentation to smooth paste of maize/sorghum at 2 and 4% (w/w) garlic or ginger singly and in combination (ginger-garlic), which resulted into 7 conditions. The samples were labeled A-H as follows: A: control samples (without garlic/ginger); B: Ogi + 2% Garlic; C: Ogi + 4% Garlic; D: Ogi + 2% Ginger; E: Ogi + 4% Ginger; F: Ogi +2% Garlic + 2% Ginger; G: Ogi +2% Garlic + 4% Ginger; H: Ogi + 4% Garlic + 2% Ginger. The slurry was evenly homogenized, then allowed to ferment spontaneously (naturally) at ambient temperature (27± 2°C) for 24 h. After fermentation the water was pressed in muslin cloth to form an ogi cake17.\n\nOgi cakes were dried for 48 h at 42± 2°C with a cabinet dryer (Gallenkamp, UK), which was then grounded into flour, cooled for 5 min at room temperature, then packaged in a pouch and sealed. The packaged samples were stored at room temperature for 16 weeks during which samples were obtained for analysis at 4 week intervals (monthly). Ogi flour samples were then placed on a shelf for further analysis18.\n\nThe total titratable acidity (TTA) of ogi flour was determined for all samples to quantify the acid produced during sample storage. 1g ogi flour was reconstituted in 10 ml of distilled water. Three drops of phenolphthalein was added as indicator; then titrated against 0.1M NaOH while gently swirling the content in the conical flask until pink colour appeared. Each ml of 0.1N NaOH used was equivalent to 90.08 mg of lactic acid. Titration readings were taken in triplicate and mean values of the readings were calculated. Total titratable acidity of lactic acid (g/ml) was calculated. A pH meter (Corning Scholar 425, UL Laboratories, Shenzhen, China) was used to determine pH values of the 5g of reconstituted flour in 50 ml of distilled water. Buffer at pH 4.0 and 7.0 were used to calibrate the pH meter. The pH of all the samples were read after stabilization of the value on the apparatus screen, the pH values were recorded in triplicate and mean values of the reading was calculated19.\n\nMoisture content determination. 5 g of each sample was weighed in triplicate into pre-weighed moisture content cans. The samples were dried for 3 h at 105 °C in the Gallenkamp hot-air oven (Gallenkamp, UK) and the weight was taken. The drying continued until their weights were constant. The samples were cooled to room temperature in a desiccator and weighed. The final weight of each sample was determined19. The moisture content was calculated from weight loss equation below\n\nMoisture content = w1-w2w1×100 (%)\n\nw1= Weight of sample before drying (g)\n\nw2= Weight of sample after drying (g)\n\nCrude protein determination. 2 g of ogi sample was weighed into a digestion flask. Kjeltec catalyst 31835-2501AE (0.8 g) and 15 ml of concentrated sulphuric acid was added to each flask. Each flask was heated on pre heated digester set (K12, Behr LaborTechnik, Germany) at 420 °C in a fume cupboard, and digested until a clear homogenous mixture was obtained. After digestion, the flask was removed from the heater, cooled, and the content was diluted with 50 ml of distilled water. The flask was then placed in micro-kjedahl analyser (Kjelmaster K-375, Buchi, Switzerland) where it received 50 ml of NaOH automatically. The mixture was subsequently heated up to release ammonia which was distilled into a conical flask containing 25 ml of 2% (w/v) boric acid as an indicator for 4 min, the ammonia reacted with boric acid to form ammonium borate which was titrated against 0.1M hydrochloric (HCl) acid until the purplish – grey end point was attained. The percentage nitrogen content of the samples was calculated using the equation below:\n\nNitrogen = A×M×0.014weightofsample(g)×100 (% g)\n\nwhere A= 0.1 HCl (ml)\n\nCrude protein content was estimated by multiplying with the factor 6.25 (The protein content in food is estimated by multiplying the determined nitrogen content by a nitrogen-to-protein conversion factor 6.25 as the standard. AOAC, 2010). The experiment was carried out in triplicate and the means for each sample were recorded\n\nCrude fat content determination. Fat content of all the ogi samples was determined by a continuous extraction liquid – solid method using soxhlet extractor with a reflux condenser and a distillation flask (E914, Buchi, Switzerland). Each sample (2 g) was weighed into a fat free thimble plugged with cotton wool and placed in the appropriate chamber of the extractor. The distillation flask was filled to two third capacities with n-hexane (60–80 boiling points); the flask was boiled on a heating mantle; the distillate was collected. Thereafter, n-hexane was recovered into a clean container until almost all had been distilled. The remaining solvent in the mixture was evaporated in a Gallenkamp hot-air oven (Gallenkamp, UK) set at 70 °C. The flask was allowed to cool subsequently in a desiccator (PYREX, Corning, Inc USA after which the final weight of the flask was determined. The difference in the final and initial weight of the distillation flask represented the oil extracted from the sample19.\n\nThe percentage of crude fat was obtained using the equation below:\n\nFat = Finalweightofflask-initialweightofflaskweightofsample(g)×100 (%)\n\nThe experiment was carried out in triplicate for each sample.\n\nCrude fibre determination. The crude fibre was determined using the weighed samples resulting from fat extraction. Each sample was transferred into conical flask and 100 ml boiling 1.25% H2SO4 added. Each beaker was heated for 30 min with periodical rotation to prevent adherence of solids to the sides of the beakers. The solution was filtered using Whatman No.1 filter paper (28413923) and rinsed with 50 ml portions boiling water; repeated trice then dried. Boiling 1.25% (w/v) NaOH solution (200 ml) was added and the mixture was boiled for 30 min after which the contents of each beaker was removed and filtered; washed with 25 ml boiling 1% sulphuric acid, three portions of 50 ml boiling water and 25 ml ethanol. The residue was dried at 100 °C to a constant weight followed by cooling in a desiccator at room temperature and weighed. The weighed residue was ignited at 600 °C in a Gallenkamp muffle furnace (Gallenkamp, UK) for 30 min, cooled in a desiccator and reweighed19.\n\nThe percentage crude fibre in each sample was calculated as:\n\nCrude fibre = w2-w3w1×100 (%)\n\nW1 = Weight of sample (g)\n\nW2 = Weight of crucible + sample (g)\n\nW3 = Weight of crucible + Ash (g)\n\nThe experiment was carried out in triplicate for each sample and the average calculated for each sample.\n\nTotal ash determination. The total ash (inorganic residue from the incineration of organic matter) was determined by dry ashing procedure. The samples (2 g) were weighed into a preweighed dry porcelain crucible. The samples were incinerated in a Gallenkamp muffle furnace (Gallenkamp, UK) at 550 °C for 6 h. After ashing, the remains were removed from the furnace, cooled to room temperature in a desiccator and weighed19. The porcelain crucible was weighed and the % total ash weight was obtained by using the equation below:\n\nTotal ash = weightofash(g)weightofsample×100 (%)\n\nCarbohydrate determination. The carbohydrate content was determined by difference. The sum of the moisture, ash, crude fiber, fat and protein of the respective samples was subtracted from 100 to obtain percentage carbohydrate19.\n\nThe amount of minerals present in the sample was determined as described by AOAC19. The ash of the sample obtained was dissolved in 10ml of 2M HNO3 and boiled for 5 min, filtered through Whatman No.1 filter paper into volumetric flask. The filtrate was made up with distilled water to 50 ml and used for determination of minerals content. Zinc (Zn), Manganese (Mn), Magnesium (Mg) and Iron (Fe) were determined by using Atomic Absorption Spectrophotometer (AAS 220GF, Buck). The standard curve for each mineral was prepared from known concentrations of mineral and the mineral content of the samples was estimated from the standard curve, while sodium and potassium content were determined using Jenway flame photometer PFP7 (Cole-palmer, UK).\n\nTotal phenolic content was determined using the modified procedure from Olaniran et al.16. Extracts (0.1 ml) of ogi flour samples were pippeted into 5.9 ml distilled water; afterward, 1.0 ml Folin Ciocalteau reagent was added to 1.0 ml of the diluted extract in test tubes. The mixture was left for 5 min before addition of 2 ml of 20% (w/v) Na2CO3. After 30 min of rigorous mixing was done with a vortex mixer, absorbance was taken at 725 nm using a spectrophotometer (Model SP9, PyeUnican UK). The results were expressed as Gallic acid equivalent (GAE) using a calibration curve with Gallic acid as standard (100 mg/ ml) y = 0.0022x - 0.0292, R² = 0.9962.\n\nThe free radical scavenging ability of ogi flour extracts using α, diphenyl-β-picrylhydrazyl (DPPH) were carried out following Pownall et al.20. 1 mL of 0.3mM DPPH dissolved in ethanol in different test tubes was added to different concentrations of 1 mL of the aqueous extracts of each of the samples. The tubes were then shaken vigorously and allowed to stand for 30 min at room temperature in the dark. A control was also prepared as mention previously without the addition of the sample. Absorbance of the samples was measured at 517 nm using a UV-VIS spectrophotometer (Model SP9, PyeUnican UK) to record the changes. Free radical scavenging ability was expressed as 50% maximal radical inhibition concentration (DPPH IC50).\n\nThe total flavonoid content (TFC) of ogi flour extract was determined following Lamien et al.21. Ogi flour extracts (0.5 ml) had ethanol (0.5 ml), 50 μl of aluminum chloride (10%), potassium acetate (50 μl), and water (1.4 ml) added to them, and then were incubated for 30 min at room temperature. The absorbance of the reaction mixture was read at 415 nm using spectrophotometer (Model SP9, PyeUnican UK). 0.01 g quercetin dissolved in 20 ml of ethanol was used to prepared standard quercetin solutions (y = 0.001x + 0.0018, R² = 0.992). The quantity of flavonoids present in the extracts was expressed as quercetin equivalent (QE). The quercetin solution without sample solution was used as positive control due to it’s a polyphenol content. All determinations were carried out in triplicate.\n\nThe means were calculated and separated using MS Excel 2010 and SAS 9.4 version (2014) respectively. Means were separated with Duncan Multiple Range Test (DMRT) at 5% level of probability.\n\n\nResults and discussions\n\nThe total titratable acidity and pH values of all biopreserved ogi flour samples with only 2% garlic, 4% garlic, 4% ginger and samples with blends of 2% garlic-2% ginger, 2% garlic-4% ginger and 4% garlic-2% ginger were stable throughout the 16 weeks of storage. Addition of powdered garlic and ginger improved the stability of ogi flour in terms of pH and total titratable acidity (TTA) values throughout the study of 16 weeks when compared with the control. pH and total titratable acidity of the control were stable for 8 weeks during storage (Figure 1 and Figure 2). With the exception of ogi flour (sorghum) containing 2% ginger, the pH was stable for 12 weeks followed by a slight increase from 3.75-3.88 till the end of storage. The addition of garlic and ginger slightly increased the ash content (0.04%), similar trends were observed in the protein content. However, in all biopreserved ogi samples containing garlic-ginger, a decrease in moisture content was recorded, with the lowest in ogi (sorghum) containing 2% garlic-4% ginger (7.70 %), when compared to the control sample (8.17 %). The moisture content of all biopreserved samples as presented in Table 1 and Table 2 ranged between 7.72-8.17%, and is less than the 10% recommendation for floury product as reported by Ikujenlola et al.22. The proximate composition of samples were comparable to findings reported during production of ogi flour from cereal17,23. The addition also increased most of the mineral content of ogi samples. Ogi (maize) containing blends of 4% garlic and 2% ginger had the highest amount of sodium, iron, and manganese. Ogi (sorghum) containing blends of 4% garlic and 2% ginger has the highest amount of magnesium and zinc (Table 3 and Table 4). The addition of garlic and ginger also enhanced the obtainable minerals in ogi. Garlic has been reported as rich source of minerals24. The total phenolic content (TPC) of the ogi flour without biopreservative, increased throughout the 16 weeks of storage from 144.50, to 152.63 GAE mg/g, and 171.50-185.75 GAE mg/g, for maize and sorghum respectively. Stable TPCs for the first 8 weeks of storage were observed in biopreserved ogi flour (maize) samples, which then increased up to the end of the storage period (Figure 3a and b). The total antioxidant radical scavenging activities of ogi flour (maize) without biopreservatives decreased throughout the period of storage from 1.54 to 1.85 mg/ml. However, the total antioxidant radical scavenging activities of ogi flour (sorghum) without biopreservative was stable in the first 8 weeks, and then decreased until the end of the storage period. Relatively stable total antioxidant radical scavenging activities were observed in all biopreserved ogi flour from maize throughout the period of storage (Figure 4a), while in sorghum samples a gradual increase throughout the 16 weeks of storage was recorded (Figure 4b). The total flavonoid content (TFC) of all ogi flour samples during the 16 weeks of storage ranged between 114.64-168.11 mg QUE/ 100g, and 150.70-198.83 mg QUE/ 100g in samples from maize and sorghum respectively. The total flavonoid content of all ogi flour (maize) without biopreservatives decreased throughout the period of storage from 132.01 to 114.04 mg QUE/ 100g. However, the TFC of all biopreserved samples were relatively stable throughout the 16 weeks of storage (Figures 5a and 5b). Comparing biopreserved samples the highest total flavonoid content was observed in samples containing blends of 4% garlic-2% ginger (168.11 and 198.83 mg QUE/ 100g for maize and sorghum respectively) and the lowest in samples containing only 2% ginger (140.01 and 170.44mg QUE/ 100g for maize and sorghum respectively). Relatively stable total antioxidant radical scavenging activities were observed in all biopreserved ogi flour from maize throughout the period of storage. It was observed that antioxidant radical scavenging activities of all biopreserved ogi flour samples from sorghum gradually increased throughout the 16 weeks of storage (Figure 5) However, the TFC of all biopreserved samples were relatively stable throughout the 16 weeks of storage. The results of this study showed that garlic and ginger can be considered good sources of natural compounds with significant antioxidant activity. A combination of garlic and ginger exerted a synergistic effect on the radical scavenging activities of ogi samples with the highest effect observed in samples containing 4% garlic and 2% ginger. It also increased the total flavonoid content, and enhanced its stability in flour samples during storage.\n\npH of Ogi flour made with (a) maize and (b) sorghum with Garlic and Ginger. Sample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\nTitratable acidity Ogi flour made with (a) maize and (b) sorghum with Garlic and Ginger. Sample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\nSample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\nSample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\nValues are means (n = 3) ± standard deviation. Means followed by different superscripts are significantly different (p < 0.05) along column according to Duncan multiple range test. Sample codes: A: Ogi, B: Ogi + 2% Garlic, F: Ogi +2% Garlic + 2% Ginger, G; Ogi +2% Garlic+ 4% Ginger, H: Ogi + 4% Garlic+ 2% Ginger.\n\nValues are means (n = 3) ± standard deviation. Means followed by different superscripts are significantly different (p < 0.05) along column according to Duncan multiple range test. Sample codes: A: Ogi, B: Ogi + 2% Garlic, F: Ogi +2% Garlic + 2% Ginger, G; Ogi +2% Garlic+ 4% Ginger, H: Ogi + 4% Garlic+ 2% Ginger.\n\nTotal Phenolic Content Ogi flour made with (a) maize and (b) sorghum with Garlic and Ginger. Sample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\nDPPH Radical Scavenging Activity Ogi flour made with (a) maize and (b) sorghum with Garlic or Ginger. Sample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\nTotal Flavonoid Content Ogi flour made with (a) maize and (b) sorghum with Garlic or Ginger. Sample codes: A: Ogi Flour, B: Ogi Flour + 2% Garlic, C: Ogi Flour + 4% Garlic, D: Ogi Flour + 2% Ginger, E: Ogi Flour + 4% Ginger, F: Ogi Flour +2% Garlic + 2% Ginger, G; Ogi Flour+2% Garlic + 4% Ginger, H: Ogi Flour +4% Garlic + 2% Ginger.\n\n\nConclusion\n\nThis study has shown that the proximate compositions of biopreserved ogi flour samples were relatively stable, and that the addition of garlic and ginger slightly increased mineral content during storage. The free radical scavenging activity; total phenolic and flavonoid content also increased correspondingly with the antioxidant activity of the ogi. Therefore, the addition of garlic and ginger as biopreservative into ogi flour both singly or as blends at 2 or 4 % w/w can be used, and will not negatively affect its quality. Although not well known to ogi consumers, the biopreserved ogi flours showed better nutritional values and may be administered as a health food.\n\n\nData availability\n\nUnderlying data is available from Figshare.\n\nFigshare: Dataset 1. Data for Proximate and antioxidant activities of bio-preserved ogi flour with garlic and ginger. https://doi.org/10.6084/m9.figshare.7415795.v125\n\nLicense: CC0 1.0 Universal (CC0 1.0) Public Domain Dedication", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nEshetie T: Review of Quality Protein Maize as Food and Feed: In Alleviating Protein Deficiency in Developing Countries. Am J Food Nutr. Science and Education Publishing; 2017; 5(3): 99–105. [cited 2018 Aug 15]. Publisher Full Text\n\nTenywa MM, Nyamwaro SO, Kalibwani R, et al.: Innovation Opportunities in Sorghum Production in Uganda. 2018; 2. [cited 2018 Nov 8]. Reference Source\n\nAjeigbe HA, Akinseye FM, Ayuba K, et al.: Productivity and Water Use Efficiency of Sorghum [Sorghum bicolor (L.) Moench] Grown under Different Nitrogen Applications in Sudan Savanna Zone, Nigeria. 2018; 2018: 7676058. Publisher Full Text\n\nFAO: Food and Agriculture Organization of the United Nations. 2018. [cited 2018 Nov 16]. Reference Source\n\nPontieri P, Troisi J, Di Fiore R, et al.: Mineral contents in grains of seven food-grade sorghum hybrids grown in a Mediterranean environment. AJCS. 2014; 8(11): 1550–1559. [cited 2018 Nov 8]. Reference Source\n\nKulamarva AG, Sosle VR, Raghavan GSV: Nutritional and Rheological Properties of Sorghum. Int J Food Prop. Taylor & Francis Group; 2009; 12(1): 55–69. [cited 2018 Sep 5]. Publisher Full Text\n\nRouf Shah T, Prasad K, Kumar P: Maize-A potential source of human nutrition and health: A review. Cogent Food Agric. 2016; 2: 1166995. [cited 2018 Nov 8]. Publisher Full Text\n\nSumbo A, Victor IA: Comparison of chemical composition, functional properties and amino acids composition of quality protein maize and common maize (Zea may L). Afr J Food Sci Technol. 2013; 5(3): 81–9. [cited 2018 Aug 21]. Publisher Full Text\n\nCsapó J, Schobert N: Production of a high-nutritional-value functional food, the Update1 bread, with the supplementation of the wheat flour with high-protein-content raw food materials. Acta Univ Sapientiae, Aliment. 2017; 10(1): 36–60. Publisher Full Text\n\nOmemu AM, Okafor UI, Obadina AO, et al.: Microbiological assessment of maize ogi cofermented with pigeon pea. Food Sci Nutr. Wiley-Blackwell; 2018; 6(5): 1238–53. [cited 2018 Jul 23]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVictor IA, Olubukola OB: Potential Complementary Food from Quality Protein Maize (Zea mays L.) Supplemented with Sesame (Sesamum indicum) and Mushroom (Oudemansiella radicata). J Nutr Food Sci. 2018; 8(3): 698. [cited 2018 Nov 8]. Publisher Full Text\n\nOyewole OA, Isah P: Locally Fermented Foods in Nigeria and their Significance to National Economy: a Review. J Rec Adv Agri. 2012; 1(4): 92–102. Reference Source\n\nOlaniran AF, Abiose SH, Adeniran AH: Biopreservative Effect of Ginger (Zingiber officinale) and Garlic Powder (Allium sativum) on Tomato Paste. J Food Saf. 2015; 35(4): 440–52. Publisher Full Text\n\nTende JA, Ayo JO, Mohammed A, et al.: Effect of Garlic (Allium Sativum) and Ginger (Zingiber Officinale) Extracts on Haemato-Biochemical Parameters and Liver Enzyme Activities in Wistar Rats. Int J Nutr Food Sci. 2014; 3(5): 380–6. [cited 2018 Nov 16]. Publisher Full Text\n\nFrank F, Xu Y, Jiang Q, et al.: Protective effects of garlic (Allium sativum) and ginger (Zingiber officinale) on physicochemical and microbial attributes of liquid smoked silver carp (Hypophthalmichthys molitrix) wrapped in aluminium foil during chilled storage. Afr J Food Sci. 2014; 8(1): 1–8. [cited 2018 Nov 16]. Publisher Full Text\n\nOlaniran AF, Abiose SH, Gbadamosi SO: Effect of Ginger and Garlic as Biopreservatives on Proximate Composition and Antioxidant Activity of Stored Tomato Paste. Ife J Technol. 2013; 22(1): 15–20. Reference Source\n\nFarinde EO: Title: Chemical and sensory properties of sieved and unsieved fortified “ogi”. Nat Sci. 2015; 13(1). [cited 2018 Aug 15]. Reference Source\n\nAkanbi CT, Ade Omowaye BI, Ojo A, et al.: Effect of Processing Factors on Rheological Properties of Ogi. Int J Food Prop. Taylor & Francis Group; 2003; 6(3): 405–18. [cited 2018 Aug 15]. Publisher Full Text\n\nAOAC International W, Horwitz W, Latimer GW: Official methods of analysis of AOAC International. Gaithersburg MD.: AOAC International; 2010 [cited 2018 Aug 21]. Reference Source\n\nPownall TL, Udenigwe CC, Aluko RE: Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. J Agric Food Chem. 2010; 58(8): 4712–8. [cited 2018 Nov 8]. PubMed Abstract \| Publisher Full Text\n\nScienceDirect (Online service) A, Lamien CE, Romito M, et al.: Determination of the total phenolic, flavonoid and proline Contents in Burkina Fasan Honey, as well as their radical scavenging activity. Food chem. Applied Science Publishers; 2005; 91(3): 571–577. [cited 2018 Nov 8]. Publisher Full Text\n\nVictor IA, Oguntuase SO, Vincent OS: Physico-Chemical Properties of Complementary Food from Malted Quality Protein Maize(Zea mays L.) and Defatted Fluted Pumpkin Flour (Telfairia occidentalis Hook, F). Food and Public Health. 2013; 3(6): 323–8. Reference Source\n\nOlamide A, Nathaniel T: Physical, Physico-Chemical and Chemical Properties of Two Maize Varieties (BR-9928-DMR-SY and TZL-Comp-4C2). 2015; 5(7). Reference Source\n\nUdu-Ibiam OE, Ogbu O, Ibiam UA, et al.: Phytochemical and Antioxidant Analyses of Selected Edible Mushrooms, Ginger and Garlic from Ebonyi State, Nigeria. IOSR Int J Pharm Biol Sci (IOSR-JPBS). 2014; 9(3): 86–91. [cited 2018 Nov 8]. Publisher Full Text\n\nOlaniran A, Abiose S: Data for Proximate and antioxidant activities of bio-preserved ogi flour with garlic and ginger. figshare. Paper. 2018. http://www.doi.org/10.6084/m9.figshare.7415795.v1" }	[ { "id": "45441", "date": "20 Mar 2019", "name": "Dolapo Oladiran", "expertise": [ "Reviewer Expertise Food chemistry", "Sensory Science", "Oral processing and ingestive behaviours", "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\nThe research aims to investigate the effect of the use of garlic and ginger as bio-preservatives on the proximate and antioxidant properties of ogi flour made from cereal grains (sorghum or maize). The experiment has been well designed and overall, this study contributes to the knowledge about the potential use of local spices as natural preservatives in food. Still, authors should consider the following important comments.\nThere are a few typographical errors throughout the manuscript e.g. in the last paragraph under the introduction, authors should consider ‘nutritional’ instead of ‘nutritionals’ quality of ogi…. Under methods specifically where ‘ogi preparation co-fermented with garlic and ginger’ is described, authors should decide whether to use past tense (as in winnowed, steeped) or present continuous tense (as in draining, milling).\nThe authors should consider giving further scientific depth. This can be done by expounding on the compounds present in ginger and garlic that make them good preservatives and the scientific rationale behind how these compounds exert their bio-preservative properties. If these are added, the technicality of the study especially the discussion bit would be greatly improved.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "47900", "date": "17 Jun 2019", "name": "Abimbola K. Arise", "expertise": [ "Reviewer Expertise Food chemistry", "protein chemistry", "food product development", "cereals and legume processing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract: This is well written and provides an exact overview of the work that was done.\nIntroduction: This section is comprehensive and relevant. Although the references cited are relevant\nMethodology: The choice of experimental design and method are adequate and there is a clear thread between the sections.\nResults: The results are clearly presented and adequately interpreted.\nDiscussion: The data analysis methods are adequate and result sufficiently discussed and compared to existing literature. This study is relevant and important particularly in the light of combating malnutrition in Africa especially among children because ogi is the major weaning food in Africa. The paper integrates well with the current research. The paper is generally well laid out and written.\nHowever, there is need to pay attention to tenses. Some other editorial correction have been indicated in the reviewed manuscript attached.\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/7-1936
https://f1000research.com/articles/8-1040/v1	10 Jul 19	{ "type": "Research Article", "title": "Screening of terpenoids as potential therapeutics against Zaire ebolavirus infection through pharmacophore-based drug design", "authors": [ "Ade Hanna Natalia", "Usman Sumo Friend Tambunan", "Ade Hanna Natalia" ], "abstract": "Backgroud: Ebola virus disease (EVD) has spread to various countries in the world and has caused many deaths. Five different virus species can cause EVD, but the most virulent is Zaire ebolavirus (EBOV). The genome of EBOV includes seven genes that encode proteins playing essential roles in the virus lifecycle. Among these proteins, VP24 plays a vital role in the inhibition of the host cells’ immune system. Therefore, VP24 is a potential target for EVD therapy. In the present study, a potential inhibitor of EBOV VP24 activity was identified through pharmacophore-based drug design. Methods: This research was a in silico study, using pharmacophore based molecular docking simulation to obtain inhibitor candidates. Result: Terpenoids were used as VP24 inhibitor candidates. In particular, 55,979 terpenoids were obtained from the PubChem database. An initial screening based on the toxicity prediction test was performed with DataWarrior software: 3,353 ligands were shown to have a favorable toxicity profile, but only 1,375 among them had suitable pharmacophore features. These ligands were used for pharmacophore-based rigid and flexible molecular docking simulations with PDB ID: 4M0Q, chosen as the crystal structure of EBOV VP24. Six ligands predicted to have strong molecular interactions with EBOV VP24 underwent pharmacological property analysis through various software packages, including DataWarrior, SwissADME, admetSAR, pkCSM, and Toxtree. Conclusions: Taxumairol V was identified as the best candidate for EVD drug therapy via EBOV VP24 inhibition based on its molecular properties, predicted molecular interactions with the target molecule, and predicted pharmacological properties.", "keywords": [ "Zaire ebolavirus", "viral protein 24", "terpenoid", "pharmacophore", "molecular docking simulation", "ADMET." ], "content": "Introduction\n\nThe Ebola virus disease (EVD) is an often-lethal hemorrhagic fever for human and non-human primates. The World Health Organization (WHO) has declared EVD one of the global public health threats. EVD is caused by various Ebola virus species that are members of the Filoviridae family, some of which have spread to countries like the United Kingdom and the United States1. Five Ebola virus species cause Ebola disease, namely Bundibugyo, Reston, Zaire, Tai Forest, and Sudan2. The Zaire ebolavirus (EBOV) is the most virulent among them, with a mortality rate of up to 90%, and is responsible for all EVD outbreaks that have occurred worldwide since 1976, mainly in Gabon, the Democratic Republic of the Congo, and West Africa1. The symptoms of EVD include myalgia, fever, decreased plasma volume, anorexia, hypotension, diarrhea, and mucosal bleeding in the genitourinary and gastrointestinal tracts2.\n\nEBOV’s genome includes seven genes that encode as many proteins that play essential roles in the lifecycle of the virus. The seven genes are known to encode the viral proteins (VP) 24, VP30, VP35, VP40, nucleoprotein (NP), glycoprotein (GP), and L (polymerase)1. Of importance amongst these, VP24 is responsible for the maturation of viral nucleocapsids and the regulation of viral transcription and replication. VP24 and VP40 have important roles in virus budding and assembling, the VP24 and NP have a contribution for viral budding and releasing3. VP24 also has a key role in the inhibition of the host cells’ immune system, especially regarding interferon signaling. VP24 is able to bind karyopherin alpha, which prevents the accumulation of tyrosine-phosphorylated STAT1 (pSTAT) in the nucleus. Afterward, the binding of IFN-stimulated response elements (ISREs) does not occur and inhibits the interferon signaling system and causes inhibition of host-mediated IFN responses as the body's defense against the Ebola virus4. Therefore, VP24 is a potential drug target in the treatment of EVD5.\n\nUntil now, no drugs have been developed to treat EVD, and potential candidates are still in the development stage. Computer-aided drug discovery and development (CADDD) is an efficient approach to identifying compounds characterized by high affinity and selectivity for macromolecular targets and beneficial pharmacokinetic and pharmacodynamic properties6. Use of CADDD enables the screening and prediction of the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) of drug candidates for drug discovery and development7. Therefore, with CADDD we were able to quickly eliminate drug candidates with low biological activity and unsatisfactory ADME-Tox properties, avoiding the implementation of the time-consuming subsequent steps of the analysis7. The pharmacophore features test in CADDD enables identification of molecular species that are likely to display a high inhibitory affinity and interaction with macromolecules recognized as important targets for disease treatment8. The site of the pharmacophore is indicated with vectors and colored spheres that denote the atom’s characteristics: hydrophobic, hydrogen-bond acceptor, hydrogen-bond donor, aromatic, cationic, or anionic9. Pharmacophore features are an advanced tool in drug discovery to screen drug candidates, optimize lead compounds, and design new compounds. Additionally, they are commonly used as templates to screen molecules (hits) with similar chemical features8.\n\nTerpenoids are a group of compounds that we investigated as inhibitors of EBOV VP24 via computational pharmacophore-based drug design. Terpenoid compounds are the largest group of natural products, and they are built starting from isoprene units and synthesized through the mevalonate and non-mevalonate pathway10. Members of this compound family are classified according to the number of isoprene units that make up their structures; namely, hemiterpenoids comprise one isoprene unit and five carbon atoms, monoterpenoids are composed of two isoprene units and ten carbon atoms. Sesquiterpenoids have three isoprene units and fifteen carbon atoms, diterpenoids have four isoprene units and twenty carbon atoms, sesterterpenoids have five isoprene units and twenty-five carbon atoms, triterpenoids have six isoprene units and thirty carbon atoms, tetraterpenoids have eight isoprene units and forty carbon atoms, and polyterpenoids have more than forty carbon atoms and more than eight isoprene units10. Terpenoids exhibit unique pharmacological and biological activities, including antibacterial11, anti-inflammatory12, anticancer13, and antiviral14 activities.\n\n\nMethods\n\nThis study was performed employing a CADDD approach using the pharmacophore feature to identify pharmacological properties and inhibitory activity toward EBOV VP24. Various online and offline software packages, such as Molecular Operating Environment 2014.09 (MOE), Toxtree v2.6.13, DataWarrior v04.07.02, AdmetSAR, pkCSM, and SwissADME, were used to analyze the pharmacological properties of all ligands and to obtain the best drug candidates for anti-EVD therapy15,16. Figure 1 presents the research pipeline of this study.\n\nThe 3D crystal structure of EBOV VP24 was obtained from the RCSB Protein Data Bank —PDB ID: 4M0Q by Edwards et al.17. Data on 4M0Q were saved in .pdb format, and the file thus obtained was then opened in MOE 2014.09 for protein preparation and optimization. In this study, preparation and optimization of 4M0Q was performed using the LigX features in MOE 2014.09 with the Amber 10: EHT and solvation of the gas phase. Notably, AMBER10: EHT has been chosen as the forcefield for simulations involving large biomolecules, like proteins and nucleic acids18. Then, the geometry of the protein crystal was optimized, and the energy of the protein was minimized. Finally, the optimized structure of EBOV VP24 was stored in .moe format.\n\nTerpenoids were obtained from the PubChem database. They are classified as organic chemicals in the chemicals and drugs category. In the organic chemicals category, terpenoids belong to the hydrocarbon group. Data from terpenoids from several subclasses, namely cannabinoids, carotenoids, diterpenes, dolichol, gefarnates, hemiterpenes, monoterpenes, polyisoprenyl phosphates, sesquiterpenes, sesterterpenes, and triterpenes, were collected. The structures of 55,979 terpenoids were saved in .sdf format.\n\nThe mentioned structures were evaluated performing the toxicity prediction test using DataWarrior v04.07.02 to identify ligands with favorable characteristics for molecular docking. Subsequently, the ligands underwent preparation and optimization with MOE 2014.09 software using modified MMFF94x as a force field and gas phase solvation. The standard ligands in this stage of the study were gossypetin and bisdemethoxycurcumin which were used to compare the characteristics of the best drug candidates for treating the EVD19. Thus, the structures of the ligands selected were saved in .mdb format.\n\nIn drug discovery, the determination of pharmacophore features contributes to the identification of ligands that have the potential to achieve a specific desired effect on a target molecule. The pharmacophore features of EBOV VP24 were obtained through molecular docking simulations with standard ligands. The standard ligands were made to dock using the site finder feature, which enabled us to identify the binding pocket of 4M0Q/EBOV VP24. Then, the molecular interaction of standard ligands with EBOV VP24 was used to determine the protein’s pharmacophore features through the pharmacophore query editor in the MOE 2014.09 software with gas phase solvation and Amber10: EHT as a force field. Finally, the pharmacophore features of 4M0Q/EBOV VP24 were saved in .ph4 format20.\n\nA two-step molecular docking simulation was performed: rigid docking followed by flexible docking. The rigid docking process refers to the condition whereby the protein is regarded as rigid and the ligand as dynamic. Therefore, the ligand will find the most stable conformation with the protein target without the latter undergoing any changes in bond angles or bond lengths. On the other hand, in the flexible docking protocol, the conformational change that the binding site of the protein target undergoes upon docking is also taken into consideration. Ligand and protein are regarded as flexible in this case, and they will seek the conformation of the most stable ligand-protein complex to optimize the strength of the bonds between ligand and receptor21. In this study, the rigid and flexible molecular docking simulations were used alongside the pharmacophore features to dock the ligands to 4M0Q. Molecular docking simulations were performed using MOE 2014.09 software with gas phase solvation and AMBER 10: EHT as a force field.\n\nTwo types of scoring functions were used in our study, namely London dG and GBVI/WSA dG. The London dG scoring function provides analytical data on hydrophobic, ionic, and hydrogen-bonding interactions and affords an estimate of the free binding energy of ligand-receptor bonds based on the given conformations22. The GBVI/WSA dG scoring function enables researchers to determine the value of refinement parameters that will afford an estimate of the ligand binding energy in each pose. Moreover, the GBVI/WSA dG scoring function displays high sensitivity to the modification of the protein environment. Therefore, docking accuracy and the strength of protein-ligand bonds can be influenced by the GBVI/WSA dG scoring function23. Based on the results of molecular docking simulations, ligands were dropped from further analysis when they displayed values for Gibbs free binding energy (∆Gbinding) that were smaller than those of the standard ligands and when the value of the root-mean-square deviation (RMSD) of the ligands was above 2.0 Å.\n\nThe best ligands based on the results of the previous analysis underwent pharmacological property analysis. Various online and offline software packages were used to screen the ADME-Tox properties of the ligands, such as DataWarrior, Toxtree, SwissADME, admetSAR, and pkCSM. DataWarrior was used to analyze the ligand’s mutagenicity, drug-likeness, and tumorigenicity. Toxtree was used to evaluate the carcinogenicity and mutagenicity of the ligands24. SwissADME was utilized to predict ligand’s pharmacokinetics and physicochemical properties25. Finally, the absorption, distribution, metabolism, and excretion properties of the ligands were predicted with admetSAR and pkCSM26.\n\n\nResults and discussion\n\nVP24 is an essential protein in EBOV’s lifecycle. Therefore, VP24 is a potential target when trying to inhibit EBOV replication and prevent EVD progression. The protein with PDB ID: 4M0Q was chosen as EBOV VP24 crystal structure, based on the fact that this structure has also been used in other studies19. The structure of PDB ID: 4M0Q was obtained through X-ray diffraction with a resolution of 1.921 Å. The protein’s active site comprises several amino acid residues, namely Y41, G44, I45, E46, F47, D48, R154, S151, S155, L158, S225, T226, F230, and T23127. In this study, all non-essential molecules in EBOV VP24 structure with PDB ID: 4M0Q, such as water, were eliminated, because their presence in the crystal structure can affect the interactions between ligand and protein28. The protonate 3D step was used for the addition of hydrogen atoms to the protein structure to mimic the hydrogen atoms in protein structure that may occur in nature15.\n\nIn this study, 55,979 terpenoids were selected in the PubChem database as potential inhibitors of EBOV VP24. The ligands were screened using DataWarrior v04.07.02 to identify disqualifying properties, such as mutagenicity, tumorigenicity, having negative reproductive effects, and being an irritant, and to recognize positive traits like drug-likeness. In fact, the DataWarrior software can be used to predict reproductive effects as well as the mutagenic, irritant, and tumorigenic properties of a particular compound based on its constituent fragments. Drug candidates displaying similar or identical structural traits as compounds known to be an irritant, upon contact, can cause the human body to display dryness or reddening29. In the DataWarrior software package, benzanthrone derivatives, silicon ether, and halogenated phenol ethers are regarded as irritant structural alerts whose use can lead to irritation in the human body30. The tumorigenicity of drug candidates is another important trait to identify because tumorigenic compounds can induce tumor cell proliferation in human tissues31.\n\nCompounds predicted to pose a risk due to their mutagenic characteristics may cause transmissible genetic damage, including gene mutations, chromosomal aberrations, and changes in the number of chromosomes32. Nitroaromatic compounds, nitrobenzene derivatives, and phenylamine derivatives are regarded as mutagenic structural alerts by DataWarrior33. In the present study, gossypetin was predicted to be mutagenic because of its 5,8-dihydroxychromone fragment. Based on toxicity predictions made using DataWarrior, 3,353 ligands were identified as possessing the desirable characteristics to inhibit EBOV VP24 activity. The ligands’ geometries were optimized to obtain ligands having the features that can be using the MMFF94x force field with gas phase solvation15.\n\nPharmacophore features play a vital role in determining the optimal molecular interactions between ligand and target macromolecule that trigger or block a particular biological activity of the macromolecule. Pharmacophore features are identified through ligand-based or structure-based construction. In the ligand-based construction, the pharmacophore feature is determined by collecting a set of ligands which shows biological activity against a particular target macromolecule and extracting the chemical properties that confer upon the molecules their bioactivity. In the structure-based construction, the pharmacophore feature is determined via analysis of the possible interaction sites between ligands and macromolecular targets. Thus, pharmacophore features identified can be used in de novo design, virtual screening, or for the optimization of lead compounds. In pharmacophore-based virtual screening, a particular pharmacophore feature is used as a screening template to find molecules that have a similar feature as the template (hits)8. Therefore, based on similarities with the pharmacophore feature used as a template, the hits are expected to display the desired biological activity.\n\nIn this study, two pharmacophore features were generated based on the interaction between the standard ligands (bisdemethoxycurcumin and gossypetin) and the protein target (4M0Q EBOV VP24)19,34. As shown from the structures of the ligand-target molecule complexes depicted in Figure 2, the hydrogen acceptor (acc) feature, reported in blue color, was constructed based on the interaction between standard ligands and the side chain of arginine B154. The hydrogen donor feature, reported in purple, was generated in reference to the interaction between the standard ligands and the side chain of aspartic acid A48. The pharmacophore features of EBOV VP24 were analyzed and validated using standard ligands. Finally, 1,375 hit ligands that match the pharmacophore features mentioned above were identified so that they could be used in molecular docking simulations. The 4M0Q EBOV VP24 protein along with the pharmacophore features is presented in Figure 2.\n\nPharmacophore features of Zaire ebolavirus protein VP24 (PDB ID: 4M0Q) identified by interaction with ligands (A) bisdemethoxycurcumin and (B) gossypetin located in the viral protein’s binding pocket\n\nIn this study, molecular docking simulations were performed to assess the protein-ligand binding affinity and predict the conformations of ligand and macromolecular target in the protein-ligand complex35. All of the 1,375 hit ligands identified in the previous stage of the procedure underwent a molecular docking simulation through pharmacophore-based rigid docking. In particular, a retain value of 30 was used to identify hits in the first simulation, and a retain value of 100 was used for the same purpose in the second simulation with Amber10: EHT as a forcefield and gas phase as the solvation parameter for both simulations. Of the 1,375 ligands whose structures were subjected to the pharmacophore-based rigid docking simulation, 855 were considered satisfactory as the values assigned to the relevant simulation at a minimum matched the retained value of 30. Then, these hit ligands were screened based on their RMSD value (<2 Å), leading to the selection of 651 ligands. These 651 ligands were then used in a pharmacophore-based rigid molecular docking simulation using a retain value of 100, leading to the selection of 642 ligands. Then, these ligands were screened based on their RMSD value, leading to the selection of 581 ligands. These 581 ligands were subjected to pharmacophore-based flexible docking simulations utilizing a retain value of 100. The 577 ligands were selected, and their RMSD values were evaluated, leading to the selection of 344 ligands, which were analyzed based on their ∆Gbinding value, RMSD value, and interaction with the 4M0Q EBOV VP24 protein. In particular, ligands were selected if the value of their ∆Gbinding was lower than the corresponding parameters calculated for the standard ligands, indicating that the relevant protein-ligand conformation was more stable than that involving the standard ligands and 4M0Q15. Rigid docking show the best performance rates between 50-75%, while flexible docking methods can enhance pose prediction up to 80–95%36.\n\nAdditionally, ligands were selected if their RMSD value was below 2.0 Å since a value of this magnitude indicates that the protein and ligand conformations produced by the molecular docking simulation are highly accurate37. In this study, the ability of ligands to inhibit the activity of the target protein was predicted through the pKi value. A large, positive value for pKi indicates that the protein-ligand complex has a low dissociation rate38. Then, all ligands were screened based on their interaction with 4M0Q. Ultimately, six ligands were selected as the best candidates for the inhibition of 4M0Q activity, namely 3-O-acetyluncaric acid, carnosic acid, ilexgenin A, negundoside tasumatrol G, and taxumairol V, which are characterized by advantageous traits regarding ∆Gbinding, RMSD value, and strength of the protein-ligand interaction.\n\nAmong these six inhibitor candidates, there are three ligands which fulfill the topological polar surface area (TPSA) value (in excess of 140 Å)39. Increased permeation rate are correlates to decreased polar surface area40. Carnosic acid displayed the lowest RMSD value among the six selected terpenoids, although standard ligand gossypetin had an even lower RMSD value than carnosic acid. Negundoside had the lowest ∆Gbinding value and the highest pKi value, indicating its superior ability to inhibit the activity of EBOV VP24. Table 1 summarizes the molecular properties of the six ligands just discussed and of the two standard ligands.\n\nStandard ligand; RMSD: root-mean-square deviation; MW: molecular weight.\n\nIn this study, the molecular interactions between the target protein and ligand identified by molecular docking simulations were analyzed to quantify the ability of each ligand to interact with the target protein. All ligands were found to interact with the target protein’s active site. Ilexgenin A displayed the highest number of molecular interactions with the target protein; it displayed interactions with six active-site amino acid residues of EBOV VP24, namely glycine B44, isoleucine B45, glutamine B46, aspartic acid A48, arginine A154, and phenylalanine A230. Negundoside is the second-best ligand in terms of the number of interactions with the target protein. This ligand is involved in hydrogen-binding interactions with five amino acid residues in the protein’s active site: lysine A39, glutamic acid A46, aspartic acid A48, arginine A154, and phenylalanine A230.\n\nTasumatrol G is involved in three hydrogen-binding interactions with amino acid residues in the protein’s active site: aspartic acid A48, arginine A154, and alanine A229. Taxumairol V and carnosic acid are each involved in three hydrogen-binding interactions with EBOV VP24. Taxumairol V is involved in hydrogen-binding interactions with three amino acid residues located in the active site of EBOV VP24, namely aspartic acid A48, arginine A154, and arginine B154. Meanwhile, the carnosic acid showed have hydrogen-bonding interactions with aspartic acid A48, arginine A154, and arginine B154. The 3-O-acetyluncaric acid showed the lowest number of hydrogen-binding interactions with the target protein. Table 2 depicts the structures of selected simulated ligand–4M0Q complexes and the interactions that characterize them.\n\nStandard ligand.\n\nPharmacological tests were carried out to predict the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) properties of the six selected terpenoid ligands, and to identify the ligand among them with the potential to be used as a drug in EVD therapy (full data for the final 6 ligands is available as underlying data41. The prediction of ADME-Tox properties was performed using various software packages because each of them evaluates different traits in the pharmacological tests. In this study, the pharmacological tests were performed with software packages Toxtree, swissADME, pkCSM, and admetSAR.\n\nToxtree analysis was implemented to determine the mutagenic potential regarding Salmonella typhimurium TA100, potential carcinogenic based on the quantitative structure–activity relationship (QSAR), genotoxic carcinogenicity, and non-genotoxic carcinogenicity of the ligands. Table 3 summarizes the results of the pharmacological tests performed with Toxtree. According to this analysis, 3-O-acetyluncaric acid, taxumairol V, ilexgenin A, and negundoside were not potential carcinogenic, mutagenic, genotoxic carcinogenic, and non-genotoxic carcinogenic compounds. Carnosic acid exhibited the characteristics of a non-genotoxic carcinogen and a mutagen. Genotoxic carcinogens can interact directly with DNA, thus, producing DNA adducts and DNA damage. Non-genotoxic carcinogens, on the other hand, do not interact directly with DNA, but they can damage cellular structures and change cell proliferation rates42. The Toxtree software comprises several structural alerts for non-genotoxic carcinogens, including the presence of thiocarbonyl, halogenated benzene, and halogenated dibenzodioxin groups43. Carnosic acid and tasumatrol G exhibited the characteristics of non-genotoxic carcinogens because they include n-alkyl carboxylic acid and thiourea groups within their structure, which match the software’s thiocarbonyl structural alert. Thiourea is a non-genotoxic carcinogen that can cause cancer in the thyroid gland. In fact, thiourea acts as a peroxidase inhibitor in the thyroid gland, inhibiting thyroxine production. A reduced synthesis level of the thyroxine hormone causes, in turn, the pituitary gland to increase thyrotropin hormone secretion, resulting in an increased risk of thyroid cancer44.\n\nstandard ligand\n\nIn this study, the potential mutagenicity of ligands was determined using the Toxtree and admetSAR software packages through the Ames test and potential S. typhimurium TA 100 mutagenicity test. In this test, chemicals are analyzed in terms of their mutagenicity toward S. typhimurium, given that bacteria belonging to this species are highly sensitive to mutagenic compounds. In particular, in Ames tests, S. typhimurium bacteria modified so that they cannot synthesize histidine are exposed to potentially mutagenic compounds. These compounds may in turn cause mutations in the bacterial his-operon, a type of change that would be made evident by the ability of the modified bacteria to grow in culture media lacking histidine. In the prediction of the mutagenic properties of ligands, the Toxtree software packages comprise a structural alert for mutagenic compounds based on results from Ames tests; structures that set off this alert include aromatic amines and nitroarenes45.\n\nGiven that carnosic acid comprises an aromatic amine group, Toxtree-based analysis classified this ligand as potentially mutagenic. In fact, aromatic amines will undergo biotransformation in the human body that results in the formation of covalent bonds with guanine, which may, in turn, cause DNA mutations. Therefore, carnosic acid was removed from the list of potential drug candidates for EVD therapy. Meanwhile, based on the Ames test which was performed using admetSAR software, all the best ligands showed no Ames toxic, which means that it did not have the potential to be mutagenic compound46. Importantly, the results of mutagenicity analysis performed using Toxtree and admetSAR were different from each other because the two software packages use different databases to formulate predictions on the mutagenic potential of ligands.\n\nThe six EBOV VP24 inhibitor candidates and the two standard ligands underwent analysis for potential mutagenicity, acute oral toxicity, human intestinal absorption (HIA), and cytochrome P450 (CYP)-inhibiting activity using the admetSAR software package. Table 4 reports data on the relevant pharmacological properties. HIA is an important oral bioavailability indicator47, and it is given a positive (+) value when a compound is promptly absorbed in the human intestine, or a negative (-) one, when the compound has poor absorption characteristics in the human intestine. The only negundoside displayed a negative HIA value following analysis, meaning that this ligand has poor or weak absorptivity in the human intestine. Regarding acute oral toxicity, compounds subjected to analysis may fall into four categories: category I designates highly toxic compounds, category II designates moderately toxic compounds, category III designates compounds of low toxicity, and category IV designates non-toxic compounds48. Acute oral toxicity analysis performed with the admetSAR software package led to ilexgenine A, negundoside, and tasumatrol G being classified as highly toxic. 3-O-acetyluncaric acid, taxumairol V, and carnosic acid were instead classified as low-toxicity compounds. Ilexgenine A and negundoside were, thus, removed from the list of potential candidates for the inhibition of EBOV VP24 activity. Trasumatrol G was retained for analysis given its good molecular docking characteristics.\n\nNote: Standard ligand, CYP: cytochrome P450; HIA: human intestinal absorption.\n\nIt is essential to identify CYP-inhibiting properties to develop a new drug. In fact, CYPs belong to an enzyme family that plays a key role in the metabolism of drugs, as its members catalyze the biotransformation of drugs in advance of their decomposition and excretion49. The AdmetSAR software package can be used to analyze the ability of ligands to act as inhibitors of several CYP isoforms that play a role in drug metabolism, namely 1A2, 2C9, 2C19, 2D6, and 3A4. In this study, carnosic acid, bisdemethoxycurcumin, and gossypetin displayed the potential to inhibit CYP 1A2 activity. Carnosic acid and bisdemethoxycurcumin displayed the potential of inhibiting the activity of CYP 2C9 and CYP 2C19. Bisdemethoxycurcumin and gossypetin displayed the potential to inhibit CYP 3A4 activity. Drugs that have the potential to inhibit the activity of various CYP isoforms may cause undesirable side effects, as they may cause drug metabolic disorders and the accumulation of potentially toxic drug metabolites50. Therefore, it was concluded that carnosic acid, gossypetin, and bisdemethoxycurcumin were not suitable candidates for the development of drugs for EVD therapy.\n\nLigands’ potential for hepatoxicity and hERG I-inhibiting activity were analyzed using the pkCSM software package. Table 5 summarizes results from this analysis. The human Ether-à-go-go Related Gene (hERG) is a selective potassium ion channel that regulates potassium ion transfer and is expressed in various cells and tissues, including cardiac myocytes, neurons, smooth muscles, and neuroendocrine cells51. Inhibition of hERG I can extend cardiac repolarization, which may result in ventricular arrhythmias, torsades de pointes, and sudden cardiac death52. Terfenadine, cisapride, astemizole, and grepafloxacin are drugs known to have inhibitory effects on hERG. I, and they have been withdrawn from the market because they can cause arrhythmias53. None of the ligands tested were predicted to display any hERG I-inhibiting activity. Regarding the potential for hepatoxicity, carnosic acid was predicted to cause damage to the liver, an organ that plays an important role in the metabolism of proteins, fats, carbohydrates, and toxins54. Therefore, carnosic acid was removed from the list of drug candidates for the inhibition of EBOV VP24 protein activity.\n\nNote: *Standard ligand. A yellow column head means the ligands were analyzed by pkCSM, whereas a green column head means that the ligands were analyzed by swissADME.\n\nThe predicted bioavailability and synthetic accessibility of ligands were determined through analysis performed with the swissADME software package (Table 5). Bioavailability is the fraction of a drug that can be absorbed by the body and the concentration of that drug which reach systemic circulation according to the total dose of the drug55. The bioavailability prediction score of a compound ranges from 0 to 1. If the bioavailability value of a compound is less than 0.5 (<0.5), the compound is predicted to have low bioavailability. If, on the other hand, that value is above 0.5 (> 0.5), the compound is predicted to have high bioavailability56. Based on the results of the bioavailability test, negundoside is predicted to display the lowest bioavailability (bioavailability score: 0.11). Therefore, negundoside was removed from the list of candidate drugs that inhibit VP24 Zaire ebolavirus activity.\n\nSynthetic accessibility (SA) is an important parameter in drug design because, in some cases, compounds designed computationally prove very difficult to synthesize57. The SA score ranges from a value of 1, indicating that the compound is very easy to synthesize, to a value of 10, indicating that the compound is very difficult to synthesize25. Based on the low SA score, carnosic acid is predicted to be the easiest compound to synthesize. 3-O-acetyluncaric acid and ilexgenin A have high SA scores, meaning that these ligands are predicted not to be easily synthesized.\n\nResults from the molecular docking simulations and pharmacological tests conducted in this study indicate that taxumairol V is the best drug candidate to inhibit EBOV VP24 protein activity, based on its ∆Gbinding, RMSD value, molecular weight, predicted interaction with the target protein and predicted pharmacological properties. Taxumairol V’s molecular weight is under 500 Dalton, and this ligand has a high propensity to inhibit the activity of the target protein, even though it does not have the highest pKi value among all the ligands tested. Furthermore, taxumairol V is predicted to pose no risk as a potential mutagenic, tumorigenic, carcinogenic, or irritant drug. This compound is predicted to display no inhibitory activity toward CYP, or hERG I. Taxumairol V is also predicted to display good HIA, and it is characterized by an intermediate bioavailability score. Finally, taxumairol V has an average SA score, indicating that it should be moderately easy to synthesize.\n\n\nConclusions\n\nIn silico approaches aid drug discovery and the development of chemical species that can achieve a desired biological effect. A total of 55,979 terpenoids were screened based on their potential toxicity, resulting in the identification of 3,353 compounds predicted to display beneficial properties. Six best drug candidates that have good molecular properties and are predicted to display strong molecular interaction with the EBOV VP24 protein were subsequently identified after molecular docking simulations were performed. These ligands were then subjected to pharmacological analyses performed using various online and offline software packages. Based on its molecular properties, predicted molecular interaction with the target protein, and predicted pharmacological characteristics, taxumairol V was identified as the best overall drug candidate to inhibit EBOV VP24 protein activity. Therefore, taxumairol V has the potential to be the basis for a drug to be developed to treat Zaire ebolavirus-caused Ebola disease.\n\n\nData availability\n\nEbola virus VP24 structure from Protein Data Bank, Accession number PBD:4M0Q: http://identifiers.org/pdb:4M0Q\n\nFigshare: All Test Result of Molecular Docking Simulation. https://doi.org/10.6084/m9.figshare.8275079.v141\n\nThis project contains the following underlying data:\n\nResult of Molecular Docking.xlsx (Molecular docking results)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis work was supported by the University of Indonesia’s Directorate of Research and Community Service (DRPM) [NKB-02/UN.2R3.1/HKP.05.00/2019]\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors give appreciation to the Directorate of Research and Community Engagement (DRPM) Universitas Indonesia for the experiment facilities. 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Publisher Full Text\n\nArcangeli A, Becchetti A: hERG Channels: From Antitargets to Novel Targets for Cancer Therapy. Clin Cancer Res. 2017; 23(1): 3–5. PubMed Abstract \| Publisher Full Text\n\nKratz JM, Grienke U, Scheel O, et al.: Natural products modulating the hERG channel: heartaches and hope. Nat Prod Rep. 2017; 34(8): 957–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurdagi S, Subbotina J, Lees-Miller J, et al.: Insights into the molecular mechanism of hERG1 channel activation and blockade by drugs. Curr Med Chem. 2010; 17(30): 3514–32. PubMed Abstract \| Publisher Full Text\n\nPandit A, Sachdeva T, Bafna P: Drug-Induced Hepatotoxicity: A Review. J Appl Pharm Sci. 2012; 02(05): 233–43. Reference Source\n\nAllam AN, El gamal SS, Naggar VF: Bioavailability: A Pharmaceutical Review. Int J Pharm Biotechnol. 2011; 1(1): 80–96. Reference Source\n\nKim MT, Sedykh A, Chakravarti SK, et al.: Critical evaluation of human oral bioavailability for pharmaceutical drugs by using various cheminformatics approaches. Pharm Res. 2014; 31(4): 1002–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukunishi Y, Kurosawa T, Mikami Y, et al.: Prediction of synthetic accessibility based on commercially available compound databases. J Chem Inf Model. 2014; 54(12): 3259–67. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56523", "date": "04 Dec 2019", "name": "Ndumiso N. Mhlongo", "expertise": [ "Reviewer Expertise Molecular modeling of proteins and small molecules implicated in infectious diseases", "Drug Design: Homology Modeling", "Molecular Docking & Virtual Screeningo Protein conformation and energetics", "Peptide/Protein-surface interface", "Electronic molecular structure", "X-ray reflection data interpretation by simulation" ], "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 identifies a potential EBOV VP24 inhibitor via pharmacophore-based virtual screening. Selected compounds were also assessed for safety properties using numerous in silico methods.\n\nThe work is clearly and accurately presented but it doesn't cite current literature. Only 3 literature sources were cited from 2018, and 0 citations from 2019.\n\nThe study design is partly appropriate and partly technically sound. This study is missing binding free energies extracted from molecular dynamics simulations of the reported systems. The reported docking results are generated from static crystal structures which is not a sound reflection of a biological system. The docking results must be correlated to the binding free energies from molecular dynamics simulations, which serve as a core validation of the docking results presented.\n\nThe conclusions drawn are not adequately supported by the presented results since the point raised above in 2 has not been fulfilled.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5088", "date": "10 Dec 2019", "name": "Usman Sumo Friend Tambunan", "role": "Author Response", "response": "We thank Ndumiso N. Mhlongo for his review and suggestion.We want to respond regarding his comment:1. The writing process of this article was finished at the end of 2018, and the submission process took place in early of 2019. this explain why this article does not contain any relevant reference from 2019 publication.2. We will add the data for the molecular dynamics simulation to explain further about the interaction, as the validation for the molecular docking simulation result." } ] }, { "id": "70796", "date": "30 Sep 2020", "name": "Daryono Hadi Tjahjono", "expertise": [ "Reviewer Expertise Medicinal chemistry", "molecular modeling and drug design." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting enough and has described the screening of terpenoids as potential therapeutics against Zaire ebolavirus infection through pharmacophore-based drug design. However, it not enough to screen molecules just using only a molecular docking.\nAuthors are suggested to improve (major revision) the manuscript, i.e:\nupdate the references by citing the current literature;\n\nconduct MD simulation for the best six molecules, and please analyze in detail the binding energy (as well as its components), residues involved in the interaction, hydrogen bond occupancy, etc.\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?\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/8-1040
https://f1000research.com/articles/8-1025/v1	09 Jul 19	{ "type": "Opinion Article", "title": "Public health research needs for molecular epidemiology and to emphasize homeostasis – could the omnipotent endopeptidase inhibitor α-2-macroglobulin be a meaningful biomarker?", "authors": [ "Frank Peter Schelp", "Ratthaphol Kraiklang", "Benja Muktabhant", "Pornpimon Chupanit", "Pattara Sanchaisuriya", "Ratthaphol Kraiklang", "Benja Muktabhant", "Pornpimon Chupanit", "Pattara Sanchaisuriya" ], "abstract": "Public health authorities in low- and middle-income countries face dramatic challenges in handling rapidly increasing non-communicable diseases (NCDs), due to the epidemiological- and particularly nutritional transition. Among major reasons for the development of NCDs are smoking and alcohol, but overnutrition and obesity are also major threats to population health. Obesity is related to diabetes and cancer, but also has a genetic background. It is difficult to recommend a healthy nutrition. This is because of conflicting nutritional conceptions, and given the complexity of human metabolism understanding this topic can be difficult for the laymen. Public health measures advocating physical activity and refraining from high intake of energy, sugar and soft drinks need to be enhanced by supporting the ‘intrinsic motivation’ to preserve a good health. The mission of public health should be to increase awareness about the complexity of human metabolism, and the involvement of genetic and epigenetics in health and diseases. To maintain homeostasis, means to keep an optimal relationship between catabolism and synthesis, seems to be of particular interest. Preconditions for this is, that public health institutions within the administration- and academic sector follow up developments in life science and molecular biology and conduct population-based research making use of molecular epidemiology, especially those related to key metabolic steps and maintenance of ‘homeostasis’, in balancing catabolism and anabolism. A prospective biomarker for this situation might be α-2-macroglobulin.", "keywords": [ "Public health", "non-communicable diseases", "sustainable development goals", "biomarkers", "dietary restriction", "homeostasis", "metabolic syndrome", "alpha-2-macroglobulin" ], "content": "Background\n\nNon-communicable diseases (NCDs), such as cardiovascular diseases, diabetes, chronic obstructive pulmonary diseases and cancer, are the main focus of the Sustainable Development Goals (SDGs)1. Target 3.4 of the SDGs intends to decrease premature death through NCDs by one third up to 2030. The four main risk factors mentioned are smoking, ‘unhealthy’ diets, physical inactivity and ‘harmful’ use of alcohol. Governments and public health authorities are encouraged to enforce so called 16 ‘best buy’ strategies to reach the target. While it is well accepted that tobacco smoking and alcohol abuse are harmful for health and there is general agreement not to smoke and at least to refrain from too much or excessive alcohol consumption, campaigning against ‘unhealthy’ diets is more problematic. This is because of the complexity of how nutrition relates to health. The ‘global obesity pandemic’2 not only is caused by a surplus of energy in the diet, but it’s link to diabetes3 and cancer4,5 and other diseases is driven by complex metabolic pathways6–9. Not only does overnutrition play a role in the development of cardiovascular diseases, cancer, diabetes and influences aging, but the risk for obesity is also related to genetic factors10,11. Through epigenetic pathways under- and overnutrition of pregnant females might result in diabetes, cancer and cardiovascular diseases in their adult children12–15. Within the field of nutritional sciences matters are further complicated by recent developments of contradictory nutritional conceptions. There seems to be disagreement about what a healthy and appropriate diet should be and controversial opinions are justified by supportive investigations into molecular factors from both sides when either a ‘low fat high carbohydrate-‘ or a ‘low-carbohydrate, high fat diet’ are promoted16.\n\n\nThe role of molecular epidemiology in public health actions against overnutrition\n\nGovernmental authorities and public health institution, overseeing the wellbeing of the population, cannot capitulate and stop promoting ‘healthy’ nutrition, in view of the constraints. So far major public health tools for working against NCDs, in regard to nutrition, are encouragement of physical activity, trying to influence behavior and practice through sophisticated methods of social sciences17, and to increase taxes on harmful products such as sugar and soft drinks with the intention to reduce consumption18. Benefit and drawbacks of these methods are not questioned here, but it has been argued by Slattery (2002) that ‘while working at the population level of exploration, molecular epidemiology must incorporate knowledge from many disciplines to obtain an understanding of the organism, the system and the cell. Translating complex disease pathways into relevant public health messages should be the goal and the result of the art of epidemiology’19. Far too often interesting results within the field of genetics, insight into metabolic pathways and molecular components, such as enzymes and cytokines, escape the intention of those working in public health. This is plausible, as investigations using laboratory models, worms and rodents, are by no means research tools for public health. Studies exploring epigenetic effects, such as DNA methylation, as recently conducted in Estonia20 are most probably not feasible for large scale epidemiological studies in low- but also middle- and high middle income countries. However, low- and middle-income countries need to make use of advances in public health. In particular low-, but especially high middle-, income countries should have the means to follow up developments in molecular epidemiology in order to have a deeper understanding of the nature of NCDs, and should conduct as much population-based research as possible, including the use of promising biomarkers to give an insight into genetic and metabolic pathways.\n\nIn fact, besides anthropometry measurements, a number of clinical laboratory methods and biomarkers are already in common use and in future, epigenetic and molecular epidemiology could be additional suitable aspirants for population-based studies19,21,22. However, multiple constraints in the use of biomarkers, as outlined for metabolic syndrome (MetS) should be considered.\n\n\nPros and cons of biomarkers – the examples of the components of the metabolic syndrome\n\nIn assessing the nutritional status using the body mass index (BMI) and other measurements as independent variables, MetS is frequently used as dependent variable since it incorporates a number of factors related directly or indirectly to NCDs. Variables associated with the syndrome include elevated blood glucose, dyslipidemia, abdominal obesity and high blood pressure. There are five different definitions of MetS, with different thresholds of its components. Because of this ,and other controversial arguments, attempts have failed to agree on either version23. As a compromise the use of the so called ‘harmonized version’ of MetS has been recommended24. It is now considered that study participants categorized as exposed to MetS should be selected if they display three or more of the five criteria. However, this results in arbitrary groupings of individuals belonging to the ‘MetS group’, and individuals are integrated with one or two factors of MetS but less than three into the ‘non-MetS’ group. An example of using the ‘harmonized version’ MetS version in grouping study participants into the MetS- and the non-MetS group is given in Table 2 of a recent publication25. To apply the ‘harmonized version’ weakens the validity of MetS as a dependent variable. The ‘nature’ of MetS as a ‘syndrome’ is also questioned. The term ‘syndrome’ should be used in case ‘the whole is greater than the parts’26, but this is doubtful27 and before selecting MetS as independent variable it should be considered that a number of factors influence MetS such as age, sex and ethnicity.\n\n\nBiomarkers representing key factor for the metabolism\n\nAs mentioned above, the use of MetS seems to be problematic and the recommendation of ‘waist circumference’ as a strong indicator of obesity and ‘insulin resistance’ as one of the metabolic key factors, could be a worthwhile alternatives for MetS24. Waste circumference is a good measurement for overnutrition, because energy intake in excess is stored in the abdominal fat tissue. The adipokines of the fat tissue, by excreting inflammatory molecules, increase the risk to develop diabetes and cancer. Insulin resistance is ‘the intersection’ either for the way to health or to metabolic disturbances. The absence of a general standard for waist circumference, however, is a disadvantage, and waist circumference needs to be standardized for different population groups28, but with the homeostatic model assessment (HOMA) on hand, a method is available for estimating insulin resistance in epidemiological studies29.\n\nTrying to find and test biomarkers mirroring key metabolic steps for health and disease should be one major objective for molecular epidemiology. It is equally important to gain insight into the interaction of anabolism with catabolism. A candidate for the latter aspect is α-2-macroglobulin (α2M). The importance of α2M has been mentioned by Ohlsson 1972, stating that ‘α2M’ may have a key role in the body’s protection against autodigestion (cited by Schelp FP et al.30), which implies that a complete deficiency will not be compatible with life. Within human plasma α2M is the largest non-immunoglobulin, and an almost omnipotent inhibitor of endopeptidases with a unique way to deactivate proteinases31. The inhibitor is found in all mammals, and the biological significance for growth and differentiation can be judged by its presence during embryogenesis, pregnancy, childhood and in aging32,33. A comprehensive overview about the molecular structure of α2M, mechanism of action, function and pathophysiology is given in the review article from Rehman et al. (2013)34.\n\n\nα-2-Macroglobulin in health and disease\n\nBesides the attention molecular biology has given to α2M, in clinical settings the inhibitor was found to be related to the development of Alzheimer’s35. Low α2M levels were observed in some patients with lung diseases36, and in advanced prostate cancer37, while in breast-38 and bladder cancer39 elevated levels of α2M were observed. It has been hypothesized that induced increase of endogenous proteinase inhibitors is protective against cancer40. The assumption, among others, was based on the ‘fat-related-cancer’ hypothesis41,42, and the low risk of vegetarians for cardiovascular diseases and cancer43, as well as the finding that α2M concentrations and other proteinase inhibitors were higher in Thai vegetarians compared with omnivores44. It was argued that the balance between proteinases and their inhibitors are regulators of tumor growth. The role of proteinase inhibitors in connection with cancer protection considered a number of different inhibitors, and the specific role of α2M remains vague since α2M is incorporated in normal but not in tumor cells. In laboratory mice alpha macroglobulin is active through ‘biomediators’ but not through its inhibitory capacity45. So far it was concluded that α2M has a role in controlling normal but not malignant growth46,47.\n\n\nThe role of α-2-macroglobulin in maintaining homeostasis in ‘dietary restriction’\n\nObesity, as outlined above, is detrimental for health but ‘dietary restriction’, defined as ‘reduced food intake by avoiding malnutrition’, extends life span in animals and humans48–50. It has been demonstrated recently, by using a mouse model, that epigenetic modification in dietary restriction delayed aging and changed the gene expressions of the lipid profile51. ‘Subclinical undernutrition’ in preschool children could reflect ‘dietary restriction without malnutrition’. The two forms of the condition are ‘wasting’, a deficit in weight for height and ‘stunting’, a deficit in height for age, adjusted to a standard52. Children categorized as wasting and stunting are apparently healthy without clinical signs of undernutrition. A number of investigation in Bangkok and villages in rural Thailand disclosed elevated α2M levels and low 3-methylhistidine (3MH) urine excretion in healthy, age matched, village children in comparison to their Bangkok counterparts53. 3MH is supposed to reflect muscle breakdown54,55. A similar result was obtained when comparing normal nourished preschool children with those deficient in weight for height. α2M serum concentration in the marginal nourished children increased over their well-nourished village counterparts56. In an animal experiment with laboratory growing rats under a marginal diet, with altered protein and energy content, serum proteinase inhibitors increased and 3HM decreased57. The results of the investigations support the hypothesis that in the situation of ‘dietary restriction’ proteinase inhibitors, including α2M, decrease muscle catabolism, and in the case of the village children kept them healthy though ‘an optimal relationship between catabolism and synthesis, thus resulting in stunting’58. In case ‘marginal nutritional intake’, results in elevated α2M serum concentrations, as expected, overweight and obese Thai adults in Bangkok, showed lower α2M serum levels compared with normal individuals. The proteinase inhibitor selected as dependent variables in a multiple regression, resulted in a model including age, sex HDL cholesterol and BMI59. A similar result was obtained studying hard working male construction laborers, in that a negative correlation were found for the variables age, weight, height, BMI, arm- and midarm circumference, triceps skinfold and HDL with α2M as the dependent variable60. α2M of female construction workers did relate to any of the variables investigated. A dietary survey conducted with apparently health Thai farmers found a statistically significant negative correlation of α2M with energy, protein, fat and carbohydrate intake61. All the results obtained from the variety of different studies seem to be in accordance with the assumption that α2M supports homeostasis in situations of a ‘challenged’ nutritional status.\n\n\nα-2-Macroglobulin in protein-energy-malnutrition\n\nAll the results obtained from the variety of different studies, as reviewed here, seem to be in accordance with the notion that α2M supports homeostasis in situations of a ‘challenged’ nutritional status and suggest that proteinase inhibitors play a key role in maintaining the metabolism in balance. This also was assumed in observing patients suffering from clinical protein-energy malnutrition (PEM). The situation in PEM children is different from subclinical malnutrition. While increased proteinase inhibitors in wasting and stunting children somehow delay catabolism to maintain homeostasis, proteinase inhibitors increasing in seriously malnourished children, interrupt the mobilization of endogenous proteins which are needed to maintain homeostasis, for instance by providing essential amino acids. The increase of proteinase inhibitors aimed to counteract the proteases released in the course of infection, turn marasmus patients to develop clinical symptoms of kwashiorkor30. Comparing marasmus with kwashiorkor, the latter is the more serious condition.\n\n\nDifferent levels of homeostasis\n\n‘Homeostasis’, as a metabolic condition depends on age, sex, genetic and environmental factors, and is maintained on different levels. This has been pointed out by Pontzer (2015) using the example of energy expenditure and energy balance62,63. This message from an evolutionary anthropologist is particularly important for public health in relation to physical activity. It is known that to lose weight by exercising has its limits. Increasing physical activity will lead to weight loss, however, excessive physical activity will not increase weight loss with increased energy expenditure; instead energy is acquired by using available resources used for basic functions of the organism under normal conditions. The consequence of this can be deadly in untrained individuals partaking in extreme physical activity e.g. a long run over 40 km. Winners of international marathon events are usually of African descent, whose ‘homeostasis’ allows them to cover the marathon distance in about two hours, while the rest of the field, using the rest of the day to finish the run. Homeostasis obviously can be achieved on different levels. The metabolism of the marathon winner allows them to economize energy expenditure much more efficiently as compared to other participants. In the regulation of the delicate balance of efficient energy expenditure, α2M might play a key role and might help to better understand the mechanisms regulating total energy expenditure. Recently a genetic hint towards regulating endurance and fatigue of muscles has been described in a rodent model64. Research in this direction might be a further step to allow a better understanding of important metabolic pathways related to energy expenditure and endurance.\n\nOther important issues for public health are also waiting for further exploration, such as how to distinguish biological- from chronological age65,66 and whether ‘dietary restriction’ is ‘beneficial’ for health and if so, are there drawbacks to be observed under certain circumstances and different ages. So for instance higher α2M levels in connection with some biological substances called ‘metallothioneins’ are beneficial in young adults but might have a harmful role in aging32.\n\n\nConclusion\n\nIn view of the challenge of non-communicable diseases, the aim of those caring for the health of the population should support the ‘intrinsic motivation’ of individuals to remain healthy. This might not only be achieved by encouraging physical activity, and restrain from smoking, alcohol and overeating. Motivation to change ‘bad’ behavior and maintaining ‘good behavior’, requires understanding what ‘bad’ behavior is meant to be, and what ‘homeostasis’ means and how to maintain it. The nature of NCDs are not yet well understood. This hampers the formulation of clear recommendations for ‘healthy’ behavior and limits the trust of the general public in health messages. Public health authorities at least should try to follow up developments and assess the significance of what is increasingly becoming known about our metabolism. Last but not least, public health should contribute to translate findings in life science, by conducting research applying molecular epidemiology, in such a way that these findings are relevant for human population groups, and may even validate α2M as a meaningful biomarker.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Grant information\n\nThis work was supported by the Research Division of Khon Kaen University under the Excellence Centre for the Research Group on Prevention and Control of Diabetes Mellitus in the Northeast of Thailand.\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\nNugent R, Bertram MY, Jan S, et al.: Investing in non-communicable disease prevention and management to advance the Sustainable Development Goals. Lancet. 2018; 391(10134): 2029–2035. 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PLoS Med. 2014; 11(2): e1001606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFradin D, Bougnères P: T2DM: Why Epigenetics? J Nutr Metab. 2011; 2011: 647514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWild CP: Complementing the genome with an \"exposome\": the outstanding challenge of environmental exposure measurement in molecular epidemiology. Cancer Epidemiol Biomar Prev. 2005; 14(8): 1847–50. PubMed Abstract \| Publisher Full Text\n\nO'Neill S, O'Driscoll L: Metabolic syndrome: a closer look at the growing epidemic and its associated pathologies. Obes Rev. 2015; 16(1): 1–12. PubMed Abstract \| Publisher Full Text\n\nAlberti KG, Eckel RH, Grundy SM, et al.: Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation. 2009; 120(16): 1640–5. PubMed Abstract \| Publisher Full Text\n\nChupanit P, Muktabhant B, Schelp FP: Dietary patterns and their association with the components of metabolic syndrome: A cross-sectional study of adults from northeast Thailand [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Res. 2018; 7: 905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpellman CW, Chemitiganti RRV: Metabolic syndrome: More questions than answers? J Am Osteophatic Association. 2010; 110(Supplement 3): eS18–eS22. Reference Source\n\nEddy DM, Schlessinger L, Heikes K: The metabolic syndrome and cardiovascular risk: implications for clinical practice. Int J Obes (Lond). 2008; 32 Suppl 2 : S5–10. PubMed Abstract \| Publisher Full Text\n\nHuxley R, Mendis S, Zheleznyakov E, et al.: Body mass index, waist circumference and waist:hip ratio as predictors of cardiovascular risk--a review of the literature. Eur J Clin Nutr. 2010; 64(1): 16–22. PubMed Abstract \| Publisher Full Text\n\nSong Y, Manson JE, Tinker L, et al.: Insulin sensitivity and insulin secretion determined by homeostasis model assessment and risk of diabetes in a multiethnic cohort of women: the Women's Health Initiative Observational Study. Diabetes Care. 2007; 30(7): 1747–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchelp FP, Migasena P, Pongpaew P, et al.: Are proteinase inhibitors a factor for the derangement of homoeostasis in protein-energy malnutrition? Am J Clin Nutr. 1978; 31(3): 451–6. PubMed Abstract \| Publisher Full Text\n\nSottrup-Jensen L: Alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem. 1989; 264(20): 11539–42. PubMed Abstract\n\nMocchegiani EG, Giacconi R, Muti E, et al.: Zinc-binding proteins (metallothionein and α-2 macroglobulin) as potential biological markers of immunosenescence. In: Straub REM, E., editor. NeuroImmune Biology. Amsterdam: Elsevier; 2004; 4: 23–40. Publisher Full Text\n\nTayade C, Esadeg S, Fang Y, et al.: Functions of alpha 2 macroglobulins in pregnancy. Mol Cell Endocrinol. 2005; 245(1–2): 60–6. PubMed Abstract \| Publisher Full Text\n\nRehman AA, Ahsan H, Khan FH: α-2-Macroglobulin: a physiological guardian. J Cell Physiol. 2013; 228(8): 1665–75. PubMed Abstract \| Publisher Full Text\n\nVarma VR, Varma S, An Y, et al.: Alpha-2 macroglobulin in Alzheimer's disease: a marker of neuronal injury through the RCAN1 pathway. Mol Psychiatry. 2017; 22(1): 13–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrüger U: [Chronic obstructive lung disease and alpha-2-macroglobulin deficiency in serum--case report]. Pneumologie. 1993; 47(9): 531–4. PubMed Abstract\n\nOhtani H, Saito M, Koshiba K: Alpha-2-macroglobulin deficiency in patients with advanced prostate cancer. Oncology. 1985; 42(6): 341–4. PubMed Abstract \| Publisher Full Text\n\nVasishta A, Baker PR, Preece PE, et al.: Serum and tissue proteinase-like peptidase activities in women undergoing total mastectomy for breast cancer. Eur J Cancer Clin Oncol. 1984; 20(2): 203–8. PubMed Abstract \| Publisher Full Text\n\nLemańska-Perek A, Lis-Kuberka J, Lepczynski A, et al.: Potential plasma biomarkers of bladder cancer identified by proteomic analysis: A pilot study. Adv Clin Exp Med. 2019; 28(3): 339–346. PubMed Abstract \| Publisher Full Text\n\nSchelp FP, Pongpaew P: Protection against cancer through nutritionally-induced increase of endogenous proteinase inhibitors--a hypothesis. Int J Epidemiol. 1988; 17(2): 287–92. PubMed Abstract \| Publisher Full Text\n\nCarroll KK: Experimental evidence of dietary factors and hormone-dependent cancers. Cancer Res. 1975; 35(11 Pt. 2): 3374–83. PubMed Abstract\n\nWynder EL: Nutrition and cancer. Fed Proc. 1976; 35(6): 1309–15. PubMed Abstract\n\nDwyer JT: Health aspects of vegetarian diets. Am J Clin Nutr. 1988; 48(3 Suppl): 712–38. PubMed Abstract \| Publisher Full Text\n\nPongpaew P, Boonyakarnkula N, Schelp FP, et al.: Serum concentrations of alpha-2-macroglobulin and other serum proteinase inhibitors in Thai vegetarians and omnivores. Nutr Res. 1994; 14(3): 337–345. Publisher Full Text\n\nKoo PH: Characterization of growth-inhibitory activities associated with an alpha-macroglobulin of mice. Cancer Res. 1982; 42(5): 1788–97. PubMed Abstract\n\nSaksela O, Wahlstrom T, Lehtovirta P, et al.: Presence of alpha 2-macroglobulin in normal but not in malignant human syncytiotrophoblasts. Cancer Res. 1981; 41(6): 2507–13. PubMed Abstract\n\nZardi L, Carnemolla B, Cagnasso D, et al.: Alpha-2-macroglobulin in normal and malignant human cells. Eur J Cancer. 1980; 16(1): 35–42. PubMed Abstract \| Publisher Full Text\n\nCava E, Fontana L: Will calorie restriction work in humans? Aging (Albany NY). 2013; 5(7): 507–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFontana L, Partridge L: Promoting health and longevity through diet: from model organisms to humans. Cell. 2015; 161(1): 106–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeindruch R, Walford RL, Fligiel S, et al.: The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J Nutr. 1986; 116(4): 641–54. PubMed Abstract \| Publisher Full Text\n\nHahn O, Grönke S, Stubbs TM, et al.: Dietary restriction protects from age-associated DNA methylation and induces epigenetic reprogramming of lipid metabolism. Genome Biol. 2017; 18(1): 56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaterlow JC, Buzina R, Keller W, et al.: The presentation and use of height and weight data for comparing the nutritional status of groups of children under the age of 10 years. Bull World Health Organ. 1977; 55(4): 489–98. PubMed Abstract \| Free Full Text\n\nPongpaew PS, Schelp FP, Vudhivai N, et al.: Alpha-2-macroglobulin, 3-methylhistidine and other biochemical parameters in preschool children of marginal nutritional status. Some evidence of an adaptation process in subclinical protein-energy malnutrition. Nutr Res. 1988; 8(11): 1213–1221. Publisher Full Text\n\nYoung VR, Alexis SD, Baliga BS, et al.: Metabolism of administered 3-methylhistidine. Lack of muscle transfer ribonucleic acid charging and quantitative excretion as 3-methylhistidine and its N-acetyl derivative. J Biol Chem. 1972; 247(11): 3592–600. PubMed Abstract\n\nYoung VR, Munro HN: Ntau-methylhistidine (3-methylhistidine) and muscle protein turnover: an overview. Fed Proc. 1978; 37(9): 2291–300. PubMed Abstract\n\nSchelp FP, Pongpaew P, Sutjahjo SR, et al.: Proteinase inhibitors and other biochemical criteria in infants and primary schoolchildren from urban and rural environments. Br J Nutr. 1981; 45(3): 451–9. PubMed Abstract \| Publisher Full Text\n\nSarisnerand V, Schelp FP, Hiller HH, et al.: Nutritionally-induced elevation of serum proteinase inhibitors and reduced 3-methylhistidine excretion in the rat. Int J Vitam Nutr Res. 1990; 60(3): 279–87. PubMed Abstract\n\nPongpaew PS, Schelp FP: Endogeneous increase of proteinase inhibitors as a possible mechanism of adaptation and subclinical undernutrition resulting in stunting. Nutr Res. 1996; 16(11–12): 1839–1845. Publisher Full Text\n\nTungtrongchitr R, Pongpaew P, Vudhivai N, et al.: Relationship between alpha-2-macroglobulin, anthropometric parameters and lipid profiles in Thai overweight and obese in Bangkok. Nutr Res. 2003; 23(9): 1143–1152. Publisher Full Text\n\nSchelp FP, Pongpaew P, Tungtrongchitr R, et al.: Anthropometry, cholesterol, HDL and LDL in relation to alpha-2-macroglobulin in Thai construction site workers. Nutr Res. 1996; 16(7): 1153–1161. Publisher Full Text\n\nPongpaew PS, Schelp FP, Supawan MTV, et al.: Influence of dietary intake on alpha-2-macroglobulin and other biochemical parameters in healthy Thai males. Nutr Res. 1991; 11(6): 559–565. Publisher Full Text\n\nPontzer H: Constrained Total Energy Expenditure and the Evolutionary Biology of Energy Balance. Exerc Sport Sci Rev. 2015; 43(3): 110–6. PubMed Abstract \| Publisher Full Text\n\nPontzer H, Raichlen DA, Wood BM, et al.: Energy expenditure and activity among Hadza hunter-gatherers. Am J Hum Biol. 2015; 27(5): 628–37. PubMed Abstract \| Publisher Full Text\n\nOkerblom J, Fletes W, Patel HH, et al.: Human-like Cmah inactivation in mice increases running endurance and decreases muscle fatigability: implications for human evolution. Proc Biol Sci. 2018; 285(1886): pii: 20181656. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJylhävä J, Pedersen NL, Hägg S: Biological Age Predictors. EBioMedicine. 2017; 21: 29–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnderwood E: The final countdown. Science. 2015; 350(6265): 1188–90. PubMed Abstract \| Publisher Full Text" }	[ { "id": "51079", "date": "03 Sep 2019", "name": "Florian J. Schweigert", "expertise": [ "Reviewer Expertise metabolism", "micronutirent", "biomarker" ], "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 submitted manuscript is a very well written review on the possible use of a2-macroglobulin as a marker of metabolism. The manuscript describes the general need of valid and meaningful biomarkers in the context of NCDs in public health research, especially with regard to overnutrition and obesity. The manuscript covers the implication of overnutrition and obesity for public health and the need of biomarkers and suggests a2-macroglobulin as such. The main part of the manuscript describes in very much detail the current knowledge of a2M in metabolic function as an omnipotent inhibitor of endopeptidases. With regard to the topic of the manuscript the authors concentrate on the knowledge regarding a2M in energy homeostasis especially with regard to energy-protein malnutrition.\nComments:\nIn general the manuscript itself is well written and balanced. However, the abstract is by no means reflecting the manuscript. It lacks condensed information on a2M and only addresses the general aspect of public health and the need of a biomarker.\n\nIt is challenging for the reader to cite a citation with refers to another citation. It cannot be retrieved without greater efforts. See second section, page 4 of Ohlsson (1972), as cited by Schelp et al.\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": "53054", "date": "21 Nov 2019", "name": "Chaowanee Chupeerach", "expertise": [ "Reviewer Expertise Nutrigenomics", "nutritional 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 manuscript is a good explanation of the need of molecular epidemiology in the public health area. This article reported the role of alpha 2 macroglobulin in the difference of metabolic state and it might be associated to homeostasis and health status. Therefore it could be a public health concern in the future.\n\nHowever, the abstract should re-arranged to be more representative of the manuscript summary.\nFor the review body, to interpret and follow the role of alpha 2 macroglobulin, the reader might need more information about this protein in terms of molecular detail.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1025
https://f1000research.com/articles/8-1024/v1	09 Jul 19	{ "type": "Case Report", "title": "Case Report: Buccal administration of hydrogen-producing blend after a mild traumatic brain injury in a professional athlete", "authors": [ "Dejan Javorac", "Valdemar Stajer", "Sergej M. Ostojic", "Dejan Javorac", "Valdemar Stajer" ], "abstract": "Background: Sport-related mild traumatic brain injury (TBI) is a serious trauma that could impair brain function of an injured athlete. Treatment solutions for mild TBI typically concentrate on complete rest, while non-traditional therapeutic options remain largely ineffective. Molecular hydrogen (H2) is an innovative neuroprotective agent that can easily reach the brain, yet no data are available concerning its value as a first-aid intervention after a mild TBI. Case report: This case report demonstrates the efficacy and safety of a hydrogen-producing dissolving tablet administered buccally during the first 24 hours post-injury in a professional soccer player who suffered a mild TBI. The patient received a formulated dosage of hydrogen every 2 hours, with the first intervention given immediately after an initial examination (~ 15 min after the injury). The overall score for Sport Concussion Assessment Tool 2 (SCAT2), a standardized method of evaluating injured athletes for concussion, increased from 68 points (severe disruption) at baseline to 84 points (mild disruption) at 24-h follow-up. The patient reported no side effects of hydrogen intervention. Conclusions: This case has demonstrated that intensive consecutive therapy with oral transmucosal hydrogen formulation is a beneficial strategy with regard to the reduction of presence and severity of symptoms of sport-related mild TBI.", "keywords": [ "Traumatic brain injury", "Concussion", "Hydrogen", "Recovery", "Buccal administration", "Athlete" ], "content": "Introduction\n\nSport-related concussion or mild traumatic brain injury (TBI) is a perilous trauma that could impair the brain function of an injured athlete both acutely and in a long-term perspective. Up to 3.8 million sport-related concussions occur in the United States annually1, with contact sports (e.g. football, wrestling, soccer, boxing, basketball, ice hockey) accounting for the greatest number of concussions2. Common signs and symptoms of acute mild TBI include physical (e.g. headache, dizziness, nausea, vomiting, fatigue), cognitive (e.g. loss of consciousness, post-traumatic amnesia, difficulties with concentration and memory), emotional (e.g. irritability, depressed mood) and sleep-related problems (e.g. drowsiness, sleeping disturbances)3. This happens probably due to a complex cascade of neurometabolic alterations in brain neurons damaged by head acceleration-deceleration mechanical forces4, with mitochondrial dysfunction, energy metabolism disturbances, and excessive oxidative stress might play a role5. Treatment solutions for sport-related concussion typically focus on complete rest at first (including a temporary withdrawal from practice and mental activities) followed by slow return-to-play activities monitored and clearance by a physician3. Non-traditional therapies, such as dietary supplements, enteral nutrition, acupuncture, and music therapy, are being considered to combat mild TBI, yet the therapeutic options remain limited6. Molecular hydrogen (H2) has recently been set forth as an innovative neuroprotective agent7 that can easily reach hard-to-reach biocompartments8, yet no data are available concerning its value as a first-aid intervention in mild TBI. This case report demonstrates the efficacy and safety of a hydrogen-producing blend administered buccally in a professional athlete that suffered a sport-related concussion.\n\n\nCase report\n\nAn apparently healthy young male professional soccer player (age 24 years, weight 75.1 kg, height 182.5 cm, professional experience 6 years) who suffered a sport-related concussion voluntarily participated in this case study. The injury occurred during a regular training session as a head-to-head collision with another player. There was a loss of consciousness for ~ 30 sec and the patient was immediately evaluated by a health care professional who confirmed the category of an injury by a physical examination. The patient had no history of concussion or other TBI in the past 6 months and no neurological, psychiatric or other chronic conditions. Written informed consent to be treated was obtained from the patient in accordance with the Declaration of Helsinki. Treatment using therapeutic hydrogen of athletes who experience a TBI was approved by the FSPE Applied Bioenergentics Lab (University of Novi Sad, Serbia; approval number, HBCS02-2019).\n\nAt the initial examination, the patient was profiled using Sport Concussion Assessment Tool 2 (SCAT2), a standardized method of evaluating injured athletes for concussion9. The total number of concussion symptoms was 18 (out of maximal 22), with a symptom severity score of 53 (out of 132). Glasgow Coma Scale (GCS) score at baseline was 13 out of 15, corresponding to mild closed head injury. Total SCAT2 score was 68 out of 100 points, approximately 25.3% below normative values (91.08)10. In addition, the patient has shown a diminished ability to keep balance during a single-leg stance test (SLST) for non-dominant foot (total number of errors 5 out of 10).\n\nA hydrogen-producing blend was administered buccally during the first 24 hours post-injury. Hydrogen was used as an exclusive treatment (along with physical and mental rest) with the main aim to reduce symptoms and signs of concussion, and it was anticipated to accelerate the acute recovery. The patient received a formulated 700-mg tablet (producing ~ 80 mL of H2) every 2 hours throughout the monitoring period, with the first intervention given immediately after an initial examination (~ 15 min after the injury). The patient was requested to put a tablet into the mouth, preferentially between the gums and teeth, and keep it inside until full dissolution. A small quantity of tap water (~ 50 mL) has been provided for mouth rinsing to improve breaking up the tablet during each administration. Buccal administration was applied due to anticipated better bioavailability, more rapid onset of action, and decreased possible risk of vomiting, a well-known manifestation of concussion. The intervention was freely provided by HRW Natural Health Products Inc. (Drink HRW Rejuvenation, New Westminster, BC, Canada).\n\nSCAT2 profiles were obtained at every 6 hours during the first 24 hours post-injury (Figure 1). Total SCAT2 scores have risen from 68 (initial score) to 72 points at first re-evaluation period and continued to increase to 84 points at the final follow-up examination. In addition, the total number of concussion symptoms decreased to 9 at 24-h follow-up, with symptom severity score dropping to 12 (out of 132), while SLST balance test improved by 25% (total number of errors 2 out of 10 at 24-h follow-up). The patient reported no side effects of hydrogen intervention, as evaluated with an open-ended questionnaire administered at the end of each treatment period; the patient was asked if the intervention resulted in any adverse events, including oral irritation or discomfort, tingling in the mouth, dizziness, nausea or flushing.\n\n\nDiscussion\n\nThis case report implies the favorable effects of hydrogen-producing dissolving tablet administered buccally as a possible first-aid treatment to improve recovery in a professional athlete with a mild TBI. Previous animal studies have demonstrated neuroprotective effects of hydrogen against perinatal11 and ischemic brain injury12, or brain damage induced by neurosurgical intervention13. In a recent pivotal study14, TBI-challenged rats exhibited significant brain injuries that were characterized by decreased survival rate and increased blood-brain barrier permeability, brain edema, and neurological dysfunction, while hydrogen treatment ameliorated the consequences of TBI. The authors concluded that hydrogen could exert a neuroprotective effect against TBI and attenuate inflammation and/or oxidative stress14, which put forward the intervention as a possible therapeutic strategy for TBI patients. Using an innovative route of administration and a benchmark tool (SCAT2) to evaluate injured athletes for concussion, we affirmed here that oral hydrogen ameliorates the presence and severity of symptoms of sport-related mild TBI. However, hydrogen has not been compared to placebo so the degree of advantage that hydrogen may provide to standard medical procedures for concussion (e.g. physical and mental rest) remains open to question. We strongly suggest further monitoring of the efficacy and safety of oral transmucosal hydrogen in sport-related mild TBIs on a larger similar case series using randomized controlled trials.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report, including any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article. No additional source data are required.", "appendix": "Grant information\n\nThis study is supported by the Serbian Ministry of Education, Science and Technological Development (#175037); the Provincial Secretariat for Higher Education and Scientific Research (#114-451-710); and the Center for Health, Exercise and Sport Sciences, Belgrade.\n\nThe funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors wish to thank Miss Nastassya Ostojic for SCAT2 data pre-processing.\n\n\nReferences\n\nLanglois JA, Rutland-Brown W, Wald MM: The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil. 2006; 21(5): 375–378. PubMed Abstract \| Publisher Full Text\n\nMarar M, McIlvain NM, Fields SK, et al.: Epidemiology of concussions among United States high school athletes in 20 sports. Am J Sports Med. 2012; 40(4): 747–755. PubMed Abstract \| Publisher Full Text\n\nClark M, Guskiewicz K: Sport-Related Traumatic Brain Injury. In: Laskowitz D, Grant G, editors. Translational Research in Traumatic Brain Injury. Boca Raton (FL): CRC Press/Taylor and Francis Group; 2016. Chapter 2. PubMed Abstract\n\nSteenerson K, Starling AJ: Pathophysiology of Sports-Related Concussion. Neurol Clin. 2017; 35(3): 403–408. PubMed Abstract \| Publisher Full Text\n\nSignoretti S, Lazzarino G, Tavazzi B, et al.: The pathophysiology of concussion. PM R. 2011; 3(10 Suppl 2): S359–368. PubMed Abstract \| Publisher Full Text\n\nLucke-Wold BP, Logsdon AF, Nguyen L, et al.: Supplements, nutrition, and alternative therapies for the treatment of traumatic brain injury. Nutr Neurosci. 2018; 21(2): 79–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIketani M, Ohsawa I: Molecular Hydrogen as a Neuroprotective Agent. Curr Neuropharmacol. 2017; 15(2): 324–331. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOstojic SM: Targeting molecular hydrogen to mitochondria: barriers and gateways. Pharmacol Res. 2015; 94: 51–53. PubMed Abstract \| Publisher Full Text\n\nRădoi A, Poca MA, Gándara D, et al.: The Sport Concussion Assessment Tool (SCAT2) for evaluating civilian mild traumatic brain injury. A pilot normative study. PLoS One. 2019; 14(2): e0212541. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmer A, Marcinak J, Hibyan S, et al.: Normative values of major SCAT2 and SCAT3 components for a college athlete population. Appl Neuropsychol Adult. 2015; 22(2): 132–140. PubMed Abstract \| Publisher Full Text\n\nImai K, Kotani T, Tsuda H, et al.: Neuroprotective potential of molecular hydrogen against perinatal brain injury via suppression of activated microglia. Free Radic Biol Med. 2016; 91: 154–163. PubMed Abstract \| Publisher Full Text\n\nNagasaki G, Masaki Y, Horiguchi T, et al.: Neuroprotective effect of hydrogen gas on brain injury in a rat transient forebrain ischemia model. Anesth Analg. 2016; 123(3S): 199. Publisher Full Text\n\nEckermann JM, Chen W, Jadhav V, et al.: Hydrogen is neuroprotective against surgically induced brain injury. Med Gas Res. 2011; 1(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTian R, Hou Z, Hao S, et al.: Hydrogen-rich water attenuates brain damage and inflammation after traumatic brain injury in rats. Brain Res. 2016; 1637: 1–13. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56420", "date": "29 Nov 2019", "name": "Cheong Hwa Ooi", "expertise": [ "Reviewer Expertise Heat Shock Proteins and Health", "Eccentric Exercise-induced Muscle Damage", "Molecular Hydrogen and Exercise Performance" ], "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\nApart from physical and mental rest, there are limited options for clinical management of sport-related concussions or mild traumatic brain injury (TBI). This case report suggests that the ingestion of hydrogen-producing tablets may be a viable treatment to improve the recovery of the concussed footballer.\nThe neuroprotective effects of molecular hydrogen against TBI have been demonstrated in animal models, in the form of hydrogen gas inhalation or hydrogen-rich water administration. However, its efficacy in treating mild TBI in humans has not been examined. The buccal administration of the molecular hydrogen in the present case report represents an innovative immediate treatment approach to manage TBI in the field.\nThe favorable outcomes of the intervention highlight the importance of a more extensive case series, and subsequently the need to conduct controlled trials in examining the therapeutic effects of molecular hydrogen on signs and symptoms of acute mild TBI in athletes.\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": "64103", "date": "16 Jun 2020", "name": "Shohei Dobashi", "expertise": [ "Reviewer Expertise Exercise", "Oxidative stress", "Hypoxia", "Molecular hydrogen", "Sport-related concussion" ], "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\nSports-related mild traumatic brain injury (TBI) is one of social issues to solve in modern society, because the sports-related TBI increases subsequent mortality from neurodegenerative disorders, such as dementia. Given that if molecular hydrogen might easily reach injured brain area and induce some cytoprotective effects as the authors mentioned in this report, we evaluate the first-aid intervention by hydrogen administration as novel and innovative approach on the sports-related TBI. In order to potentiate the view that molecular hydrogen is beneficial for acute phase of sports-related TBI, we would like to propose the authors to consider some additional concerns as written below.\n\nBecause of a case report, we understand that this study did not set placebo control trial (i.e., physical and mental rest without hydrogen treatment). However, we believe that the representation of the typical time course change in SCAT2 score without any specific medication will strongly enhance the therapeutic value of this intervention. Another point is that SCAT2 consists several clusters of assessment items (e.g., somatic, cognitive, emotional, and sleep-fatigue symptoms), nevertheless the authors displayed just only total SCAT2 score in this study. The information about which individual factor was improved by hydrogen treatment is so important to assess its efficacy and examine the target of the treatment. Thus, we would like to propose the authors to display the time course changes not only total score but also each score of SCAT2 symptoms.\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/8-1024
https://f1000research.com/articles/8-1016/v1	08 Jul 19	{ "type": "Research Note", "title": "Toxicity of the hydroalcoholic extracts of fruit leaves from the Peruvian Amazon in Artemia salina", "authors": [ "Ana Sandoval Vergara", "Armando Ismiño Riquelme", "Armando Ismiño Riquelme" ], "abstract": "Background: Medicinal plants have been used since ancient times, receiving interest in their healing potential because of their active components. The Amazon rainforest in the east of Peru has great diversity of flora, especially monocotyledons (Cocos nucifera, Mauritia flexuosa and Coffea spp.) and dicotyledons (Theobroma cacao L. and Musa spp. The toxicity of the hydroalcoholic extract of plant leaves from the Peruvian Amazon in Artemia salina was evaluated in this study. Methods: The leaves of the plants Cocos nucifera, Mauritia flexuosa, Theobroma cacao L., Coffea sp, and Musa sp were collected in the district of Cacatachi, San Martín. Phytochemical analysis of the leaves was carried out to identify their active components. The eggs of Artemia salina were provided by the Department of Animal Physiology of Universidad Nacional de Trujillo. The hydro-alcoholic extraction was carried out via the maceration method using 300 g of leaves and 500 mL of 96% ethanol for 15 days in agitation. The solutions were taken to a vertical rotavapor to obtain dry extracts and concentrations of 10, 100 and 1000 μL/ml were prepared. To test toxicity, 10 larvae were given the extract for each plant species and concentration in triplicate. The CL50 toxicity of Artemia salina samples was classified as: ˃ 1000 μL/ml (non-toxic), 500˂CL50 ≤ 1000 (low toxicity), 100˂CL50 ≤ 500 (moderate toxicity), CL50˂100 (high toxicity). Results: It was observed that Mauritia flexuosa and Musa sp. at concentrations of 10, 100 and 1000 μL/ml have high, medium and low toxicity, respectively. However, only low toxicity was observed in Cocos nucifera, Theobroma cacao L. and Coffea sp.\nConclusions: It can be concluded that the obtained results are in accordance with other studies that examined different extracts, indicating that if a sample is non-toxic to Artemia salina, then its effects will also be similar in humans.", "keywords": [ "Toxicity", "hydro-alcoholic extract", "Peruvian Amazon", "Artemia salina" ], "content": "Introduction\n\nMedicinal plants have been used since ancient times, receiving interest in their healing potential because of their active components1,2. Some developing countries use plants in primary health care, and in 2015 it was estimated that 25.6 billion dollars were spent worldwide on plant consumption and its derivatives, expected to increase to 35.4 billion dollars by 20203,4. Due to the high consumption of plants for medicinal purposes, it is necessary to study their toxicology in order to avoid side effects.\n\nThe Amazon rainforest in the east of Peru has great diversity of flora, especially monocotyledons (Cocos nucifera, Mauritia flexuosa and Coffea spp.) and dicotyledons (Theobroma cacao L. and Musa spp.); species which are used as an alternative to medicine due to their chemical properties and active components, such as steroids, phenolic compounds, flavonoids and lactones5–7. Studies have shown that the leaf extracts of some species are toxic for the human consumption because of the combination of their chemical compounds8–11.\n\nArtemia salina is a species of shrimp belonging to the Crustaceae family. Ranging from 1 to 7 mm in size, these shrimps have a cosmopolitan distribution and live in saltwater at a temperature of 6 to 35°C12. Its biology was studied as a preliminary test for ecotoxicological investigations associated with low cost and easy handling, in addition to its potential use as a practical and sustainable method for the identification of the pharmacological potential of synthetic and natural compounds and to measure their toxicity in animals13–15. The determination of plant toxicity against Artemia salina involves only one parameter: life or death, and the absence of toxicity is an indicator that the plant can be tolerated by biological systems16. Therefore, the aim of this study was to evaluate the toxicity of the hydro-alcoholic extract of fruit leaves from the Peruvian Amazon in Artemia salina.\n\n\nMethods\n\nThe eggs (20 days old) of Artemia salina were donated by the Department of Animal Physiology of Universidad Nacional de Trujillo, washed with filtered seawater and dried in dehydrated environment and then transported (with oxygen supply). One gram of eggs was used (equivalent to 700-800 eggs), left to hatch in 5 liters of filtered sea water under fluorescent artificial light (110 watts) at a temperature of 25°C for 24 h. The eggs were fed on commercial yeast extract and kept in a glass incubation chamber with abundant oxygenation to allow them to hatch and continue their biological cycle for approximately seven days. Stage III larvae (seven days old) were used as toxicity markers due to their high sensitivity and 10 larvae were used to test each concentration (10, 100 and 1000 μg / ml).\n\nA total of 500 grams of plant samples from each species (Cocos nucifera, Mauritia flexuosa, Theobroma cacao L., Coffea sp, and Musa sp) were collected by researchers (AI and AS) in the district of Cacatachi, San Martín at 295 m above sea level and 12 km to the north of Tarapoto (6°29'40\" south latitude and 76°27'57\" west longitude). The specimens were transferred in vacuum bags that were labeled with their respective scientific names at a temperature of 37°C to the Herbarium Truxillense of Universidad Nacional de Trujillo for their identification and given a registration code.\n\nLeaves were washed with distilled water, disinfected with 96% ethanol and fragmented to an approximate size of 3mm. Extraction of the hydro-alcoholic extract was carried out by maceration with 300 g of leaves and 500 mL of 96% ethanol over 15 days, with agitation carried out using a vertical rotavapor (Scilogex RE-100), run at 70rpm for 10 minutes every four hours (between 7am and 10pm) and a dry extract dissolved in 96% ethanol was obtained.\n\nPhytochemical analysis of the leaf samples was carried out to identify their active principles according to the protocol described by Miranda et al.17. Briefly, each sample was subjected to solvents of increasing polarity, in order to obtain secondary metabolites according to their solubility, using reagents and dyes to determine the presence or absence of active components such as terpenes, flavonoids, reducing sugars, among others. The assays used to determine the presence of each type of secondary metabolite are listed in Table 1. To each test tube, 1ml of pure extract was added and reagents for each assay were added that identify secondary metabolites through color change. The results of the color change were judged by eye according to the method described by Miranda and Cuellar17 and classified as light, moderate or strong.\n\n(+): light, (++): moderate, (+++): strong.\n\nConcentrations (using filtered seawater) of 10, 100 and 1000 µg/ml were than prepared according to the protocol described by Seremet et al. 201818: 5μg of extract was diluted in 5ml of filtered seawater, equivalent to 10μg/ml, 50μg of extract was diluted in 5ml of filtered seawater, equivalent to 100μg/ml, and 500μg of extract was diluted in 5ml of filtered seawater, equivalent to 1000μg/ml. The larvae were added to a test tube containing 10ml of filtered seawater and 0.5ml of the hydroalcoholic extract. A total of 10 larvae were used for each plant species and concentration and each test was performed in triplicate. A control group of 10 larvae in 10ml of filtered seawater without extract was used. The tests were performed in the biological chemistry laboratory of the Universidad Nacional de Trujillo and larvae were observed for 24 hours before the number of live larvae were counted using a stereoscope. The lethal concentration (CL50) for Artemia salina samples was used to classify toxicity as follows: ˃ 1000 µg/ml (non-toxic), 500 ˂ CL50 ≤ 1000 (low toxicity), 100 ˂ CL50 ≤ 500 (moderate toxicity), CL50 ˂ 100 (high toxicity)19.\n\nThe toxicity percentage was calculated as follows:\n\n\n\nWhere TNA = Total number of Artemia and AA = Number of alive Artemia19.\n\nThe statistical analysis was carried out using SPSS version 23, applying the student's t-test to calculate toxicity at different concentrations, with p<0.05 considered as statistically significant.\n\nGenerally, permits are required from the Environmental Ministry to perform experiments in which animals are involved. However, a permit to investigate Artemia salina was not considered necessary because it does not appear on the International Union for the Conservation of Nature (IUCN) red list and therefore it’s investigation does not represent a danger to the environment or to human beings. All efforts were made to ameliorate any suffering to the animals following the protocol described by the Ecuadorian code of practice for the care and use of animals for scientific purposes of the Ministry of Environment of Ecuador.\n\n\nResults\n\nQualitative presence of phenolic compounds can be observed in the hydro-alcoholic extract, such as flavonoids, steroids, triterpenes, reducing sugars, lactones and tannins (Table 1)20.\n\nIn Figure 1, it can be observed that there is high toxicity (10 µg/ml), moderate (100 µg/ml) and low (1000 µg/ml) in Mauritia flexuosa and Musa sp; however, only low toxicity (1000 µg/ml) is evidenced in Cocos nucífera, Coffea sp. and Theobroma cacao L (see Underlying data)21.\n\nThe results obtained with Artemia salina indicate that both Mauritia flexuosa and Musa sp in the concentrations 10, 100 and 1000 µg/ml have low, moderate and high toxicity, respectively. However, in Cocos nucífera, Coffea sp and Theobroma cacao L., only low toxicity is observed at a concentration of 1000 µg/ml, indicating that toxicity was directly proportional to the concentration of the extract used; therefore, the highest toxicity could be related to the high content of phenolic compounds and flavonoids, among other components, in accordance with previous studies22–26. Samples with values of LD50 higher than 1,000 μg/ml were considered non-toxic, in accordance with Leite et al. 200927, and values of LD50 below 1,000 μg/ml were considered to be toxic according to previous studies28–30. The characteristic phytochemical composition of each plant can change due to various factors such as room temperature, soil, pH and origin. These environmental conditions may influence the synthesis31,32 and expression of the phytochemical components in the plant, changing their toxicity.\n\n\nConclusions\n\nHealth treatments with medicinal plants are cheap and accessible to the population; however, their indiscriminate use is a risk due to the toxicity of some compounds within the plant. For this reason, it may be useful to study plant extracts with the aim of demonstrating the therapeutic or toxic action of their active components, using Artemia salina as an evaluation method. In this context, the toxicity of hydroalcoholic extracts of fruit leaves from the Peruvian Amazon was evaluated. It can be concluded that the obtained results are in accordance with other studies that examined different extracts as reported by Simões and De Almeida33, indicating that if a sample is non-toxic to Artemia salina, then its effects will also be similar to humans.\n\n\nData availability\n\nFigshare: Toxicity percentage at different concentrations of hydro-alcoholic extract. https://doi.org/10.6084/m9.figshare.7996598.v221\n\nThis project contains the following underlying data:\n\n- Toxicity table.xlsx (spreadsheet containing raw toxicity data)\n\nFigshare: Photos for the phytochemical analysis of the fruit leaves. https://doi.org/10.6084/m9.figshare.8194580.v220\n\nThis project contains the following underlying data:\n\n- Cocos nucífera.tif (photographs of the results of the phytochemical analysis for Cocos nucífera)\n\n- Coffea Sp.tif (photograph of the results of the phytochemical analysis for Coffea Sp)\n\n- Mauritia flexuosa.tif (photograph of the results of the phytochemical analysis for Mauritia flexuosa)\n\n- Musa Sp.tif (photograph of the results of the phytochemical analysis for Musa Sp)\n\n- Theobroma cacao L.tif (photograph of the results of the phytochemical analysis for Theobroma cacao L)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGouveia-Figueira SC, Gouveia CA, Carvalho MJ, et al.: Antioxidant Capacity, Cytotoxicity and Antimycobacterial Activity of Madeira Archipelago Endemic Helichrysum Dietary and Medicinal Plants. Antioxidants (Basel). 2014; 3(4): 713–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUllah S, Rashid Khan M, Ali Shah N, et al.: Ethnomedicinal plant use value in the Lakki Marwat District of Pakistan. J Ethnopharmacol. 2014; 158 Pt A: 412–22. PubMed Abstract \| Publisher Full Text\n\nTaddei-Bringas GA, Santillana-Macedo MA, Romero-Cancio JA, et al.: [Acceptance and use of medicinal plants in family medicine]. Salud Publica Mex. 1999; 41(3): 216–220. PubMed Abstract\n\nFármacos botánicos y derivados de plantas. Mercados globales. Informe de investigación BCC BIO022G. Reference Source\n\nKrishnaiah D, Sarbatly R, Nithyanandam R: A review of the antioxidant potential of medicinal plant species. Food and Bioproducts Processing. 2011; 89(3): 217–233. Publisher Full Text\n\nPereira Freire JA, Barros KBNT, Lima LKF, et al.: Phytochemistry Profile, Nutritional Properties and Pharmacological Activities of Mauritia flexuosa. J Food Sci. 2016; 81(11): R2611–R2622. PubMed Abstract \| Publisher Full Text\n\nShah NA, Khan MR, Naz K, et al.: Antioxidant potential, DNA protection, and HPLC-DAD analysis of neglected medicinal Jurinea dolomiaea roots. Biomed Res Int. 2014; 2014: 726241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartínez M, Ocampo D, Galvis A, et al.: Actividad antibacteriana y citotoxicidad in vitro de extractos etanólicos de Bauhinia variegata L. (Fabaceae) Revista Cubana de plantas medicinales. 2011; 16(4): 313–323. Reference Source\n\nMonteiro JA, Ferreira Júnior JM, Oliveira IR, et al.: Bioactivity and Toxicity of Senna cana and Senna pendula Extracts. Biochem Res Int. 2018; 2018: 8074306. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRavi B, Ramachandra Y, Sundara S, et al.: Evaluación del potencial antihelmíntico de extractos de agallas de la hoja de Terminelia chebula. (Gaertn) Retz. (Combretaceae).2014; 6: 1–4.\n\nLeite Ade S, Dantas AF, Oliveira GL, et al.: Evaluation of toxic, cytotoxic, mutagenic, and antimutagenic activities of natural and technical cashew nut shell liquids using the Allium cepa and Artemia salina bioassays. Biomed Res Int. 2015; 2015: 626835. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorroto J, Trujillo R, De la Torre Y, et al.: Actividad antimicrobiana y toxicidad frente a Artemia salina del extracto diclorometánico de raíces de Morinda royoc L. Revista cubana de plantas medicinales. 2011; 16(1): 34–42. Reference Source\n\nÁvalos-Soto J, Treviño-Neávez JF, Verde-Star MJ, et al.: Evaluación citotóxica de los extractos etanólicos de Azadirachta indica (A. Juss) sobre diferentes líneas celulares. Revista mexicana de ciencias farmacéuticas. 2014; 45(3): 39–44. Reference Source\n\nPino OP, Jorge FJ: Ensayo de Artemia: Útil herramienta de trabajo para ecotoxicólogos y químicos de productos naturales Revista protección vegetal. 2010; 34–43. Reference Source\n\nFernández-Calienes ValdésI A, Mendiola Martínez J, Monzote Fidalgo L, et al.: Evaluación de la toxicidad de extractos de plantas cubanas con posible acción antiparasitaria utilizando larvas de Artemia salina L. Revista Cubana de Medicina Tropical. 2009; 61(3): 254–8. Reference Source\n\nSilva EMF, de Castro Nascimento RB, Barreto FS, et al.: Estudo in vitro do potencial citotóxico da Annona muricata L. Revista de Ciências Farmacêutica Básica Aplicada. 2015; 36(2): 277–283. Reference Source\n\nMiranda M, Cuellar A: Mención en Productos Naturales y Terapéuticos. Maestría en Farmacia y Bioquímica. Escuela de Post-Grado. Universidad Nacional de Trujillo. Cuba, 2003; 3–5.\n\nSeremet OC, Olaru OT, Gutu CM, et al.: Toxicity of plant extracts containing pyrrolizidine alkaloids using alternative invertebrate models. Mol Med Rep. 2018; 17(6): 7757–7763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer BN, Ferrigini NR, Putnan JE, et al.: Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 1982; 45(5): 31–34. PubMed Abstract \| Publisher Full Text\n\nSandoval A: Photos for the phytochemical analysis of the fruit leaves. 2019. http://www.doi.org/10.6084/m9.figshare.8194580.v1\n\nSandoval A: Toxicity percentage at different concentrations of hydro-alcoholic extract. 2019. http://www.doi.org/10.6084/m9.figshare.7996598.v2\n\nRavi Shankara BE, Ramachandra YL, Rajan SS, et al.: Evaluating the Anticancer Potential of Ethanolic Gall Extract of Terminalia chebula (Gaertn.) Retz. (Combretaceae). J Young Pharm. 2014; 6: 1–4. 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PubMed Abstract \| Publisher Full Text\n\nCybulski WJ, Peterjohn WT, Sullivan JH: The influence of elevated ultraviolet-B radiation (UV-B) on tissue quality and decomposition of loblolly pine (Pinus taeda L.) needles. Environ Exp Bot. 2000; 44(3): 231–241. PubMed Abstract \| Publisher Full Text\n\nShen H, Tang Y, Muraoka H, et al.: Characteristics of leaf photosynthesis and simulated individual carbon budget in Primula nutans under contrasting light and temperature conditions. J Plant Res. 2008; 121(2): 191–200. PubMed Abstract \| Publisher Full Text\n\nSimões R, Almeida S: Phytochemical study Bauhinia forficata (Fabaceae). Biota Amazônia. 2015; 5(1): 27–31. Publisher Full Text" }	[ { "id": "69691", "date": "14 Sep 2020", "name": "Gabriel Alfonso Gutierrez-Rebolledo", "expertise": [ "Reviewer Expertise Toxicological and pharmacological pre-clinical evaluation of natural and synthetic compounds" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the work clearly and accurately presented and does it cite the current literature?\nThe objective of the work is innovative and is within the publishable objectives of the journal. However, although the techniques used were the correct ones to solve or fulfill the objective of the work, there are certain details that need to be refined in how much the specificities and calculations for the toxicological evaluation. The results were widely supported with updated scientific literature and related to the objectives of the experimental work.\n\nIs the study design appropriate and does the work have academic merit?\nThe proposed experimental design was adequate for the resolution of the objective and the initial hypothesis of the work. However, the arrangement within the text and its presentation format must be improved.\n\nAre sufficient details of methods and analysis provided to allow replication by others?\n\nThere were errors when making the calculations to obtain or prepare the stock solutions that would be used to obtain the concentrations to be tested for each extract. A thorough review of the calculations is recommended.\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nAlthough the correct or adequate statistical test was chosen for the type of variables and the proposed experimental design, these results of the statistical analysis were not properly represented in the graphs and mentioned in the text.\n\nAre all the source data underlying the results available to ensure full reproducibility?\nThe work can be reproducible without any doubt; however, the description of the methods, the way to calculate the results and to present / describe them should be improved.\n\nAre the conclusions drawn adequately supported by the results?\nYes, although they must be rewritten since they were not shown in a precise and concrete way, nor do they mention whether the main objective and the initial hypothesis of the work are accepted or denied with the results obtained.\n\nAbstract: In the abstract’s background sub-section, please write a short line that mentions the importance of medicinal plant’s pre-clinical toxicological studies. This line could be placed before mentioning the work’s main objective. In this same sub-section please before describing the diverse Amazonian flora; please mention also that all these species have scientific supported biological activities, but non toxicological evaluation. Please in the methods abstract’s sub-section, correct the line in which it mentions that phytochemical analysis was carried out in the leaves, instead describe that it was made in every hydro-alcoholic leaf extract. Please in this same abstract’s sub-section avoid the extensive description of each method, this could be done in its respective section in the main text. This will leave you a lot more space to describe your findings in the abstract (the most out-standing ones). Please rearrange the methods sequence as follows: biological material, hydro-alcoholic leaf extract preparation, and finally toxicity test. In the results abstract’s sub-section please re-write the first idea and you could better mentions that these two species (Mauritia flexuosa and Musa sp.) showed a concentration-dependent effect, since the way it is described, it is understood when reading it, that the higher concentration generated less toxicity when in the graphs it is observed for both species that the concentration of 1000 microliters / milliliter generated a toxicity value above 60% (highest). Also in this same paragraph please mention for which of the three tested concentrations the other species Cocos nucifera, Theobroma cacao L. and Coffea sp. exerted a low toxicity over Artemia salina. Please correct the measurement units in which are mentioned the tested concentrations in the results abstract’s sub-section change from µL/ml to µg/ml. Finally, in the conclusion abstract’s sub-section, please re-write the whole paragraph, because as is mentioned in the authors and reviewers guidelines of this journal, conclusions shouldn’t be suppositions, instead conclusions must mention if the work’s main objective and its hypotheses were supported by the experimental findings, as well in this case you should mention of all the tested and studies vegetal species which were the less toxic, but also it should never be ensured that a substance is not toxic by having performed only one test of so many that are required in toxicological studies.\n\nIntroduction: Please before start describing the bio-diversity of Amazonian jungle and rain forest, you should add a short paragraph in which you could describe some actual examples of used medical plants that resulted toxic through scientific techniques. Fuse this new paragraph with this idea “Studies have shown that the leaf extracts of some species are toxic for the human consumption because of the combination of their chemical compounds [8-11]”. After describing the phytochemical profile of the studied species, please add information related to how they are used by the population (ethnomedicinal applications and uses), and scientific supported biological activities (the most important at least). In the last paragraph and before start describing Artemia salina experimental model importance, please add information in why is important to achieve pre-clinical toxicological studies in natural product’s evaluations, mostly in medicinal plants. Is it correct to ensure that this study in Artemia salina is sufficient to extrapolate its results to a genetically as different and remote species as the human being? If the study is carried out in Artemia salina, it is ensured that the same study should no longer be carried out or similar in other animal species?\n\nMaterials and methods: The method’s sub-section entitled: “Source and husbandry of Artemia salina”, should be rearranged after “Phytochemical analysis of plant samples” and before “Toxicity tests” sub-sections. In the method’s sub-section entitled: “Sources of plant samples”, each of the registration numbers of the herbarium specimens (vouchers), and the person in charge who carried out the taxonomic identification, should be mentioned in the writing. If the type of extract chosen was hydro-alcoholic, it should be mentioned in the sub-section entitled: \"Preparation of plant samples\", why this type of extract was chosen, as well as why it was chosen to work only with the leaves of the specimens, and also what is the water:ethanol ratio used, since the manuscript only mentions that the samples were placed by maceration in 96% alcohol. In and after the sub-title “Preparation of plant samples” please you could change plant samples for hydro-alcoholic leaf extract. Do this correction in the rest of the manuscript from here forward. If kind of studied extract was hydro-alcoholic, and then it was dried in a vertical rotary evaporator, please specify and add the temperature conditions used, since the extract has water in it, the conditions must be very specific to be able to evaporate that aqueous fraction, or it is done using a coupled vacuum pump to the rotary evaporator or with a subsequent lyophilization process, to avoid high temperatures and denature process of the metabolites present in the extract. In the “Phytochemical analysis of plant samples” sub-section, this line is confusing “Briefly, each sample was subjected to solvents of increasing polarity, in order to obtain secondary metabolites according to their solubility…”, does this mean that a chemical fractionation was carried out in a chromatography column using solvents of different polarities to group the secondary metabolites present in the extract? or that different solvents of increasing polarity were used to solubilize and restore the extract before performing the qualitative phytochemical tests? please explain better this matter. Table 1 must be located and described in the Results section, mostly as phytochemical profile results. Please change the table 1 position. Please, rearrange the “Ethical statement” position, this sub-section must be after “Phytochemical analysis of plant samples”, and before the new location of “Source and husbandry of Artemia salina” sub-section, and after this last one you could put “Toxicity tests” sub-section, followed by “Statistical analysis” sub-section closing Materials and Methods manuscript’s section. Please, in the method’s sub-section entitled: “Toxicity test”, explain please how you did the calculations to obtain the test concentrations since no matter how much I repeat mine with the data that provide in the manuscript, the final concentration it is not the same value:\n\n5µg of dried hydro-alcoholic extract + diluted in 5 ml of seawater equivalent to 10µg/ml 5µg / 5ml = 1µg/ ml Or this one calculation could be the correct one: 50µg / 5ml = 10µg/ml\n\n50 µg of dried hydro-alcoholic extract + diluted in 5 ml of seawater equivalent to 100µg/ml 50µg / 5ml = 10µg/ml Or this one calculation could be the correct one: 500µg / 5ml = 100µg/ml\n\n500µg of dried hydro-alcoholic extract + diluted in 5 ml of seawater equivalent to 1000µg/ml 500µg / 5ml = 100µg/ml Or this one calculation could be the correct one: 5000µg / 5ml = 1000µg/ml\n\nThis is an important point to correct or reassess, this does not mean that all the results obtained no longer serve, but rather readjust both their description, their discussion, conclusion and reclassify the extracts within the categories proposed in the description of the technique with the true values of the concentrations tested, which apparently were lower than those described in this version, which would be: 1, 10 and 100 µg/ml.\n\nResults: The first results that should be described in this section of the manuscript would be those related to the dry final weight of each of the hydro-alcoholic extracts, as well as the final yields of each one. Followed by the table of results of the phytochemical analysis. Place the table after the first paragraph of the results and not in the method section. In the same description of the phytochemical profile results for each specie; also please mention which one extract has more chemical complexity, due to the presence of a greater amount of metabolites and also mention of what polarity nature they are mostly. When describing your results, please mention what percentage of toxicity is related to each concentration evaluated, since it is confusing to read this section. A clear example of this reading and writing confusion is the next statement: “it can be observed that there is high toxicity (10 µg/ml), moderate (100 µg/ml) and low (1000 µg/ml) in Mauritia flexuosa and Musa sp; however, only low toxicity (1000 µg/ml) is evidenced in Cocos nucífera, Coffea sp. and Theobroma cacao L (see Underlying data)”, but how can this possible if when I see the graphic what is understood is the following: for Mauritia flexuosa toxicity percentages for each tested concentration approximately were 50% for 10µg/ml, 60% for 100µg/ml and 70% for 1000µg/ml, same behavior observed in the graphic for Musa sp., so why do you considerer 1000µg/ml as a low toxicity? In the graphic all abbreviations must be explained in the footnote, so please describe what means TA, TB, and TM. In the result’s discussion please in addition to classifying each extract according to its toxicity, relate this observed effect to what type of metabolites are present in each, and support this idea by seeking information of toxicological evaluations carried out for each family of metabolites in the same plant species, or within plants of the same scientific genre or taxonomical family. Both in the graph and described in the text when mentioning the results obtained, the significant or statistical differences between the experimental groups must be mentioned and exemplified in the graph using superscripts.\n\nConclusions: The last paragraph of this manuscript should be completely rewritten because of a conclusion in an experimental work:\n\nmust be concrete and related to the objective / hypothesis of the work.\n\nshould not be referenced, so this current paragraph of the manuscript would not go into conclusions but rather in discussions of the results.\n\nThis entire paragraph: “Health treatments with medicinal plants are cheap and accessible to the population; however, their indiscriminate use is a risk due to the toxicity of some compounds within the plant. For this reason, it may be useful to study plant extracts with the aim of demonstrating the therapeutic or toxic action of their active components, using Artemia salina as an evaluation method. In this context, the toxicity of hydroalcoholic extracts of fruit leaves from the Peruvian Amazon was evaluated. It can be concluded that the obtained results are in accordance with other studies that examined different extracts as reported by Simões and De Almeida33, indicating that if a sample is nontoxic to Artemia salina, then its effects will also be similar to humans.”, must be in the result’s discussions sub-section in the Result’s section.\n\nAs conclusions of their work, they could mention which were the main types of secondary metabolites that were found in the various extracts of the hydro-alcoholic type, as well as mention how these are related to the toxicity shown by each species, and finally, mention within the classification system used which species were most toxic.\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? I 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": [] } ]	1	https://f1000research.com/articles/8-1016
https://f1000research.com/articles/8-1012/v1	05 Jul 19	{ "type": "Research Article", "title": "Biogenetic study of the emissions of species: Pinus radiata, Eucalyptus globulus Labill and Alnus acuminata in Riobamba canton, Ecuador", "authors": [ "Benito Mendoza", "Marco Cruz", "Lucero Carrera", "Mauro Jimenez", "Juan Caicedo", "Miguel Osorio", "Luis Santillán", "Fabian Arias", "Marco Cruz", "Lucero Carrera", "Mauro Jimenez", "Juan Caicedo", "Miguel Osorio", "Luis Santillán", "Fabian Arias" ], "abstract": "Background: Air pollution is one of the biggest problems in the world, and it is generated by industrial production, vehicular flow and use of fossil fuels, leaving aside other important emission sources such as vegetation. The aim of this research is to quantify the emissions of natural volatile organic compounds produced by the forest species: Eucalyptus globulus L., Pinus radiata and Alnus acuminata in Riobamba, Ecuador. Methods: Identification of plant coverings in the years 2014 and 2017was performed using geographic information systems tools, complemented with the application of the Guenther model for the calculation of monoterpenes and other organic volatile compounds; thus, to analyze the relationship between meteorological variables and concentrations of volatile organic compounds and nitrogen dioxide per species. Results: Mathematical calculation of emissions in Riobamba showed that Eucalyptus globulus L. registered higher emissions in the years 2014-2017, followed by Pinus radiata and Alnus acuminata. These emissions are due to the vegetation cover covering each species. The analysis of volatile organic compounds in forest plantations in air is directly related to the emissions represented in the environment and correlated with the meteorological variables of temperature, global solar radiation and wind velocity. The proposed method manages to estimate concentrations of monoterpenes and volatile organic compounds for the two examined seasons, presenting the influence of the species introduced in this study such as Eucalyptus globulus L. and Pinus radiata, with a reduction in their emissions (less area found in the year 2017, with respect to 2014). However, the emission of Alnus acuminata can be quantified only in 2017, since in 2014 no records of this species were found. Conclusions: Volatile organic compound concentrations in the air are directly related to the emissions represented spatially and correlated with the meteorological variables of temperature, global solar radiation and wind velocity.", "keywords": [ "Biogenetic study", "EMISSION", "Pinus radiata", "Eucalyptus globulus Labill", "Alnus acuminata", "Riobamba canton" ], "content": "Introduction\n\nThe atmosphere contains many gases which, when presented in concentrations higher than normal, are poisonous to humans, animals and are harmful to plants; gases such as nitrogen oxides (NOx), sulphur (SOx), hydrocarbons, carbon monoxide (CO) and a wide variety of volatile organic compounds (VOCs) are considered primary pollutants, because they are emitted directly from a source. Secondary contaminants are formed by means of chemical reactions from the primary pollutants; ozone (O3) is found in this group1. In recent years, Ecuador has been more interested in emissions of natural origin, giving rise to inventories of volatile organic compounds nationwide, obtaining 1,855,600 tons/year in 20102. The Ministry of Environment, Ecuador (MEE) has developed emission inventories in the districts Ambato, Riobamba, Santo Domingo de los Colorados, Latacunga, Ibarra, Manta, Portoviejo, Esmeraldas and Milagro, giving a space to the biogenic emissions representing 3.3% of the total emissions in Riobamba3. Riobamba is located at an altitude of 2750 m above sea leave; it is in the Sierra Central region and constitutes the capital of Chimborazo4. The population of the rural areas of the Ecuadorian Highlands, including Riobamba, has been dedicated to agroforestry crops with commercial purposes5. Some of these plant species are exotic, which in addition to causing negative effects to the soil, emit polluting gases that react in the atmosphere, giving rise to the formation of new compounds that may have negative effects on humans6.\n\nIn this context, the objective of this study is to make an approximate quantification of the emissions of natural volatile organic compounds from the species Pinus radiata, Eucalyptus globulus L. and Alnus acuminata in the district, by the variation of plant coverings obtained based on spectral signatures, temperature analysis of the years 2014–2017 and application of the emission model proposed by Guenther.\n\n\nMethods\n\nBased on the area occupied by each species, plots of circular form are arranged with an area of 500 m2 each7 applying the equation of finite populations to obtain the sample size8:\n\n\n\nWhere: n represents the sample size; Z, 95% confidence level of = 1.96; N, study population; E, estimation error = 0.05; p, probability of success = 0.5; q, probability of failure = 0.5.\n\nSampling was carried out for 3 days (October 8, 9 and 10, 2018), 3750 spectral signatures of the three species under study were obtained with the Spectrum-Field Spec 4 radiometer, this in seven plots of Eucalyptus globulus L. four of Pinus radiata and four of Alnus acuminata (Table 1).\n\n*Coordinates are expressed in Universal Transverse Mercator coordinate system (Hemisphere: South; Zone: 17).\n\nThe spectral signatures were treated statistically with SAMS 3.2 software; for the correction of jumps, the Jump Correction tool was used, which corrects the level of reflectance in the signature. The spectra that were found out of the trend of the vegetative states of the small trees (those up to 20 cm in diameter) and high trees (those exceeding 20 cm in diameter) of the three species under study, were eliminated with the help of the software Minitab 18, obtaining the standard deviation grouped to rule out significant differences between the spectra grouped by plots.\n\nThe contact probe was used to analyze the spectral signatures of plants with the spectrum-radiometer Field Spec 4, selecting five samples distributed in a plot; each sample represents a tree, from which five leaf subsamples were taken from the canopy.\n\nSpectral signatures were analyzed using View Spec Pro 6.2 and Minitab 18 software; the consistency of the spectral signature reflectance levels is also statistically verified using the SAMS software, discarding those that do not present a similar trend to the metadata group.\n\nThe field assessment of the normalized difference vegetation index (NDVI) is calculated from the average wavelength between 640 to 670 nm and 850 to 880 nm, and in satellite images using bands 4 and 5 of the Landsat 8 Medi satellite to Equation 29.\n\n\n\nWhere NIR is the atmospherically corrected reflectance corresponding to the near infrared and R is the atmospherically corrected reflectance corresponding to the red.\n\nThe spectral difference reflected in the NDVI is used for the comparison of forest species coverings in the years 2014–2017, obtained by a supervised classification with the maximum likelihood classifier algorithm, using as a basis Landsat 8 satellite images5,6 with a spatial resolution of 30 x 30 m per pixel. The satellite images were obtained from the portal of the United States Geological Survey (USGS) through the Global Visualization Viewer (GloVis), making a search of the satellite images of the study area (Riobamba-Ecuador) in the years 2014 and 2017 (with a maximum cloudiness of 25%), the satellite selected was Landsat 8 due to availability and good resolution per pixel. Once selected, each of the images was downloaded separately.\n\nThe calculation and the resulting maps are made with the rasterized calculator tool of the ArcGIS 10.3 software10 (QGIS is an open-access alternative), entering the bands 4 and 5 of the satellite images, extracting the values of the index of each pixel corresponding to the monitoring plots according to the vegetative state of the species.\n\nTo obtain the result, the maximum likelihood classification algorithm11 is applied using ENVI 5.3 software. This is a comparison of the effects of the satellite image with those taken as training areas, thus assigning the pixels to the class to which they most likely belong. The resulting classification was exported to shapefile format.\n\nFor the study of temperature, data from the automatic meteorological stations of: ESPOCH, UNACH, San Juan, Alao, Tunshi, Quimiag, and Urbina were used. In addition, to determine the hourly temperature, linear regression of the form ax + b was used12. These data are interpolated with the universal kriging method13, generating hourly temperature maps for the years 2014–201720.\n\nEmissions were calculated based on the temperature schedules generated for each month. It uses the biomass density values and emission factors for monoterpenes and BVOC proposed by Guenther, described in the Underlying data. Table 1.\n\nThe time emissions of monoterpenes were calculated by means of the formulas posed by Guenther2.\n\n\n\nWhere Emon (k, time) is hourly emission of monoterpenes in each K cell (µg/h), EFjmon is standard emission factor of monoterpenes associated with J category soil use (µg/g.h), FBDj is density of foliar biomass of the J class of soil use (g/m2), Ais area of each cell (900 m2) and M(T): environmental correction factor belonging to the temperature (Equation 4).\n\n\n\nWhere: β is an empirical coefficient (0.09°K-1); T is leaf temperature (equal to environmental temperature in °K); Ts is standard temperature (303 °K),\n\nDaily emissions are obtained using Equation 5.\n\n\n\nMonthly emissions are obtained using Equation 6.\n\n\n\nThe calculation of the annual emissions of monoterpenes is obtained through Equation 7.\n\n\n\nThese are calculated with Equation 8, which was also used previously for the calculation of monoterpenes, considering the variation of emission factors2.\n\n\n\nWhere: EBVOC (k, time) is the hourly emission of BVOC in each K cell (µg/h) and EFjBVOC is the standard emission factor of BVOC associated with the J category of soil use (µg/g.h).\n\nDaily, monthly and annual emissions of other volatile organic compounds are obtained by Equation 5, Equation 6 and Equation 7, respectively.\n\nThe experimental application consisted of measurements of VOC and NO2 concentrations, using the Aeroqual S-500 gas analyzer equipment between 11:00 and 15:00, due to the higher daily temperatures occurring in this range.\n\nThe analysis of the existing correlation of the meteorological variables (temperature, solar radiation and wind velocity) with VOC concentrations was performed using Pearson's product-moment correlation coefficient14. Two-way ANOVA with post hoc Turkey’s test were used for the statistical analysis of the NO2 concentrations, grouping the variables temperature and global solar radiation. For the graphical analysis, the moving average method of order 3 was applied to obtain a smoothing of the curves14. In addition, the Pearson correlation method was used to assess the linear correlation between the VOC concentrations in the plantations of each plant species with the following variables: temperature, global solar radiation and wind speed.15. Statistical analyses were performed using Minitab v18 (Minitab, Inc.).\n\n\nResults and discussion\n\nIn the spectral signature of Eucalyptus globulus L., the reflectance level in the vegetative states (sapling and timber) does not differ significantly in the range comprising the NDVI; the highest peak of the sapling state has a reflectance of 72.18% and a timber of 72.27%15. This is due to the similarity in the structure of the leaves in the two vegetative states.\n\nThe spectral signature of Pinus radiata shows that the reflectance levels of the sapling state are slightly higher (83.82% in the highest peak), while in the timber the highest peak corresponds to 82.36% of reflectance.\n\nIn the spectral signature of Alnus acuminata, the representative spectral signature in the sapling state shows that the highest peak has 83.13% reflectance at wavelength 880 nm, which is located in the range comprising the NDVI.\n\nThe NDVI values obtained in the field are elevated values approximated to 1, being dense and healthy vegetation16. The highest value is presented in the sapling state of Alnus acuminata (0.887) and the lower result of the index corresponds to the species of Pinus radiata (0.795), whereas in the timber state, the highest value (0.808) corresponds to Eucalyptus Globulus L. and the lowest (0.819) to Pinus radiata (see Underlying data: Table 2).\n\nUsing the information from the NDVI maps of the satellite images, it was determined that in the year 2014 the minimum value (0.303) was of Pinus radiata in the timber state, and the highest in sapling state (0.622). In the year 2017, the highest value (0.537) corresponding to Pinus radiata in timber state and the lowest (0.384) is of Alnus acuminata.\n\nThe plant coverings in the years 2014–2017 for the three forest species were obtained through a supervised classification, taking advantage of the spectral difference found in the NDVI values. The reliable results were verified by means of the maximum likelihood algorithm reflected in the confusion matrix, surpassing the value of 0.85 in the Kappa coefficient, considered an almost perfect classification according to Landi and Koch17.\n\nThe variation in the area covered by forest species is important (Figure 4, Figure 5), especially in Eucalyptus globulus L., which has suffered greater deforestation, and in the last three years it has decreased 469.22 ha, and Pinus radiata has reduced 228.11 ha in the same time. Since in the year 2014 no plantations were found; in 2017 there were 44.31 ha, located mainly in the Cacha parish due to the existing deforestation programs.\n\nThe annual gross deforestation in Riobamba shows that species planted for commercial purposes, such as Pinus radiata, contributes towards 76.04 ha/year of deforestation. Eucalyptus globulus L. is deforested by 156.41 ha/year, in contrast to Alnus acuminata, the area of which has increased by 14.77 ha/year. These values represent an important part of the average annual gross deforestation in Chimborazo16, which reaches 928 ha/year.\n\nThe temperature variations with respect to time obtained from geostatistical analysis (Figure 6) shows that in the year 2017 the average hourly temperatures are slightly higher than in the year 2014, emphasizing from 13:00 to 15:00 hours, time with the highest temperatures.\n\nTemperature behaves similarly in the two years (Figure 7), but it is evident that in 2014 August is the month with the lowest average temperature, reaching 9.64°C, whereas, in 2017 July has the lowest average monthly temperature (10.01°C). The highest monthly average temperature values recorded in 2014 correspond to February (11.78°C); however, November 2017 has a higher value (12.16°C), thus, conditioning the emissions of natural VOC.\n\nMonthly emissions of monoterpenes in the year 2014 were due in a greater proportion to the species of Eucalyptus globulus L. surpassing 5.40 tons, especially in February and November (Figure 8). Pinus radiata emitted monoterpenes to a lesser amount, reaching maximum emissions of 3.03 tons in November. Alnus acuminata emissions were not recorded due to the absence of plantations.\n\nThe emissions of monoterpenes in 2014 of Eucalyptus Globulus L. correspond to 60.68 ton/year and 33.67 ton/year of Pinus radiata.\n\nMonthly emissions of BVOC in the year 2014 follow a similar annual pattern to monoterpenes. The emissions of Eucalyptus globulus L. and Pinus Radiata emit between 2.4 and 3 tons per month, reaching a maximum of 1.65 tons in November (Figure 9). The total emissions of BVOC in 2014 by Eucalyptus Globulus L. were 33.01 ton/year and for Pinus radiata were 18.32 ton/year.\n\nFor monthly emissions of monoterpenes in the year 2017, the highest emissions are generated by Eucalyptus globulus L. (Figure 10) and correspond to the month of November (4.39 tons). The lowest by Eucalyptus globulus L. occurred in July (3.59 tons). Pinus radiata emissions do not exceed 2.29 tons per month, evidencing that Alnus acuminata species presents extremely low emissions compared to the other species, reaching maximum emissions of 0.031 tons in November.\n\nTotal emissions of monoterpenes in the 2017 were reduced due to the decreased vegetal cover of each species, presenting emissions of 49.05 ton/year for Eucalyptus globulus L., 25.49 ton/year for Pinus radiata and 0.035 ton/year for Alnus acuminata; only the latter has increased, since in 2014 it was not possible to find plantations.\n\nConcerning monthly emissions of BVOC in the year 2017, the highest emissions for Eucalyptus globulus L. and Pinus Radiata occur in November (2.39 and 1.34 ton), and the lowest emissions were in July (1.95 and 1.02 ton). Emissions of Alnus acuminata have increased, reaching 0.051 tons in November and 0.41 in July, being the highest and lowest monthly emissions, respectively.\n\nThe total emissions of BVOC in 2017 in Eucalyptus Globulus L. was 26.29 ton/year and in Pinus radiata was 13.87 ton/year. These two species emit larger amounts of BVOC than Alnus acuminata, which only reached 0.571 ton/year.\n\nAccording to Pearson correlation coefficient analysis with a confidence level of 99%, VOC concentrations in plantations of Pinus radiata. have a positive significant linear correlation that is higher with temperature (R2=0.725) and global solar radiation (R2=0.535) (Figure 12), indicating that as temperature and radiation increase, VOC emissions also increase.\n\nWind velocity has a significant negative linear correlation (R2=0.528) with VOC emissions (Figure 12), i.e., when wind velocity is lower, gases tend to accumulate in the planting area, thus increasing the concentration.\n\nVOC concentrations in plantations in Eucalyptus globulus L. show a positive significant linear correlation with temperature variables (R2=0.80) and global solar radiation (R2=0.609), and a significant negative linear correlation with wind velocity (R2=-0.569) (Figure 13).\n\nThe linear relationship between VOC and meteorological variables is lower in Pinus radiata compared to Eucalyptus globulus L., demonstrating a lesser influence of the meteorological variables on VOC emissions in Pinus radiata plantations.\n\nConcentrations in the air were nil; this behavior can be related to the lower presence of existing biomass and low values of emission factors, especially monoterpenes.\n\nTwo-way ANOVA showed that the average concentrations of NO2 do not differ from each other when related to the variables of temperature and solar radiation, in plantations of Pinus radiata, Eucalyptus globulus L, and Alnus acuminata (Underlying data: Table 3).\n\nThe trend between the concentrations of NO2 and the variables temperature and global solar radiation is similar in plantations of Pinus radiata, Eucalyptus globulus L., and Alnus acuminata, demonstrating the relationship between the behavior of the climatic variables and NO2 concentrations in an environment with low anthropogenic intervention.\n\n\nConclusions\n\nThe representative spectral signatures of each species were obtained. The reflectance values were similar in the vegetative and timber states, allowing the generalization in each species, finding that the maximum level of reflectance in Eucalyptus Globulus L. is 72.2%, in Pinus radiata is 83.8% and in Alnus acuminata is 83.1%. The spectral difference found among the species allowed the obtaining of the NDVI vegetation index, which served as a basis for an optimum dissolution among classes, identifying the exact geographical location of each plant species.\n\nTemperature determines the emission of volatile organic natural compounds, particularly between the time range between 13:00 and 15:00. The spatial distribution of the temperature with respect to time indicated that biogenic emissions are concentrated in the central area of the parish, depending on the presence of forest plantations. In the year 2014, February was the month with higher temperatures reaching an average of 11.78°C, whereas, in the year 2017 the highest average temperature reached is 12.16°C in November. The lower average temperatures in 2014 was August (9.64°C) and in 2017 was July (10.01°C).\n\nEmissions of monoterpenes by Eucalyptus globulus L. in 2014 were 60.68 ton/year and were 49.05 ton/year in 2017. Emissions of BVOC were 33.01 tons in 2014 and 26.29 tons in 2017. Pinus radiata emitted 33.67 ton/year of monoterpenes in 2014 and 25.49 ton/year in 2017. Emissions of BVOC in 2014 were 18.32 ton/year, and were 13.87 ton/year in 2017. In 2017, Alnus acuminata emitted 0.035 tons/year of monoterpenes and 0.571 tons/year of BVOC. Eucalyptus globulus L. is the species with the highest emissions in both years due to the greater number of plantations, followed by Pinus radiata and Alnus acuminata. At the general level, Eucalyptus globulus L. and Pinus radiata record a decrease in emissions in 2017 when compared with 2014, which is linked to deforestation; unlike Alnus acuminata, which exhibited a small increase in plantations due to existing reforestation plans, so increased in emissions in the same way.\n\n\nData availability\n\nFigshare: Raw data for Biogenetic study of the emissions of species: Pinus radiata, Eucalyptus globulus Labill and Alnus acuminata in Riobamba canton, Ecuador, https://doi.org/10.6084/m9.figshare.8081216.v121\n\nThis project contains the following underlying data:\n\n- Spreadsheet containing raw toxicity data for biomass density and emission factors for monoterpenes and OCOV (Table 1), calculated values of the Normalized difference vegetation index (Table 2) and analysis of variance for NO2 concentrations (Table 3)\n\nFigshare: Raw data for NVDI calculation, https://doi.org/10.6084/m9.figshare.8323670.v122\n\nFigshare: Temperatures for each plot and each month, https://doi.org/10.6084/m9.figshare.8323688.v123\n\nFigshare: Monoterpenes/BVOC, https://doi.org/10.6084/m9.figshare.8323715.v124\n\nFigshare: Categories of Soil Use, https://doi.org/10.6084/m9.figshare.8323799.v125\n\nFigshare: VOC and NO2 levels for each plot, https://doi.org/10.6084/m9.figshare.8323832.v126\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nOMS: Guías para la calidad del aire. Perú. 2004. Reference Source\n\nViteri M: Estimación de las emisiones de Compuestos Orgánicos Volátiles de la vegetación del Ecuador durante el año 2010. Universidad San Francisco de Quito, Ecuador. 2012. Reference Source\n\nMinisterio del Ambiente: Inventario Preliminar de las Emisiones de Contaminantes del Aire, de los cantones Ambato, Riobamba, Santo Domingo de los Colorados, Latacunga, Ibarra, Manta, Portoviejo, Esmeraldas y Milagro. 2014; 3–124. Reference Source\n\nGADM Riobamba: Plan de Desarrollo y Ordenamiento Territorial del Cantón Riobamba. 2015.\n\nMinisterio de Agricultura Ganadería Acuacultura y Pesca [MAGAP]: Programa de Incentivos para la Reforestación con Fines Comerciales. Ecuador. 2013. Reference Source\n\nMinisterio del Ambiente: Econciencia Verde. (Primera Ed). Quito - Ecuador: Revista Especializada en Medio Ambiente. 2015; 1. Reference Source\n\nMinisterio de Agricultura Ganadería Acuacultura y Pesca [MAGAP]: Manual De Procedimientos para la Evaluación de la sobrevivencia y el mantenimiento de las plantaciones forestales comerciales. Ecuador. 2016.\n\nLópez M: Elaboración de un manual de operaciones para la captura de “firmas espectrales” en campo, validada en dos granjas experimentales. Universidad de Cuenca, Ecuador. 2014. Reference Source\n\nBravo N: Teledetección Espacial Landsat, Sentinel-2, ASTER L1T y MODIS. Perú: Universidad Nacional Agraria de la Selva, 2017. Reference Source\n\nGitas IZ, Douros K, Minakou C, et al.: Multi-temporal soil erosion risk assessment in N. Chalkidiki using a modified USLE raster model. EARSeL eProceedings. 2009; 8(1): 40–52. Reference Source\n\nRojas Espinoza G, Ortiz Iribarren O: Identificación del cilindro nudoso en imágenes TC de trozas podadas de Pinus radiata utilizando el clasificador de máxima verosimilitud. Maderas Ciencia y tecnología 2009; 11(2): 117–127. Publisher Full Text\n\nSerrano S, Zuleta D, Moscoso V, et al.: Análisis estadístico de datos meteorológicos mensuales y diarios para la determinación de variabilidad climática y cambio climático en el Distrito Metropolitano de Quito. La Granja. 2012; 16(2). Publisher Full Text\n\nVelasco E, Bernabé R: Emisiones biogénicas. (Primera Ed). México: INE-SEMARNAT. 2004. Reference Source\n\nTriola MF: Estadística. (11 Ed.). México: Pearson Educación. 2013. Reference Source\n\nGonzaga C: Aplicación de Índices de Vegetación derivados de imágenes satelitales Landsat 7 ETM + y ASTER para la caracterización de la cobertura vegetal en la zona centro de la provincia de Loja, Ecuador. Univerisdad Nacional de la Plata, Argentina. 2014. Reference Source\n\nHernández J, Montaner D: Patrones de respuesta Espectral. Lab Geomática y Ecología del Paisaje (GEP). 2009; 1–14. Reference Source\n\nCarrillo L: Determinación de la firma espectral de gynoxys sp, para la clasificación de imágenes satelitales en el bosque de ceja andina en la parroquia achupallas, cantón Alausí, provincia de Chimborazo. 2016; 144. Reference Source\n\nCarrillo L: Determinación de la firma espectral de gynoxys sp, para la clasificación de imágenes satelitales en el bosque de ceja andina en la parroquia achupallas, cantón Alausí, provincia de Chimborazo. 2016; 144. Reference Source\n\nTingey DT, Manning M, Grothaus LC, et al.: Influence of light and temperature on monoterpene emission rates from slash pine. Plant Physiol. 1980; 65(5): 797–801. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmerman D, Pavlik C, Ruggles A, et al.: An experimental comparison of ordinary and universal kriging and inverse distance weighting. Math Geol. 1999; 31(4): 375–390. Publisher Full Text\n\nMendoza B: Raw data for Biogenetic study of the emissions of species: Pinus radiata, Eucalyptus globulus Labill and Alnus acuminata in Riobamba canton, Ecuador. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8081216.v1\n\nBenito: Raw data for NVDI calculation. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8323670.v1\n\nBenito: Temperatures for each plot and each month. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8323688.v1\n\nBenito: Monoterpenes/BVOC. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8323715.v1\n\nBenito: Categories of Soil Use. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8323799.v1\n\nBenito: VOC and NO2 levels for each plot. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8323832.v1" }	[ { "id": "51402", "date": "20 Aug 2019", "name": "Mufreh S. Al-Rashidi", "expertise": [ "Reviewer Expertise Atmospheric and air pollution emission studies", "environmental impact assessment studies", "environmental engineering 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 article addresses important natural volatile organic compounds (NVOC) sources of emissions to atmosphere from a three biogenic forest species, the Eucalyptus globulus, Pinus radiata and Alnus acuminata in Riobamba, Ecuador as a case study. The research methodology is well defined using different research tools linked with each other’s to provide the results. The study conclusions are practical and inline with the results finding. However, the following minor comments and changes need to be taken into consideration by the authors:\nIt is better in the titles of the article the word “Biogenetic study” change to “ Biogenic study” as the word Biogenic signified for the emission of substance (i.e., VOC, BVOC and NO2) from a living material such the plants and/or species in this study.\n\nIn introduction, 2nd line, “as nitrogen oxides (NOx), sulphur (SOx), …” to be change to “as nitrogen oxides (NOx), sulphur oxides (SOx), …”.\n\nFigures 1, 2, 3, and 4 need to be reproduced with high resolution ones. In Figures 12 and 13, the x-axis name should be change from “COV (ppm)” to “VOC (ppm)”.\n\nMost of the references are documented research and/or study carried out in Ecuador in Spanish language, authors recommended to update the references section with the original source of references which are mostly published in English such as the Guenther model these references are as follows:\nGuenther, A., Monson, R. K., and Fall, R.: Isoprene and monoterpene emission rate variability: Observations with Eucalyptus and emission rate algorithm development, J. Geophys. Res., 96, 10799–10808, 19911.\n\nGuenther, A., Hewitt, C. N., Erickson, D., Fall, R., Geron, C., Graedel, T., Harley, P., Klinger, L., Lerdau, M., McKay, W. A., Pierce, T., Scholes, B., Steinbrecher, R., Tallamraju, R., Taylor, J., and Zimmermann, P.: A global model of natural volatile organic compound emissions, J. Geophys. Res., 100, 8873–8892, 19952.\n\nGuenther, A., Karl, T., Harley, P., Wiedinmyer, C., Palmer, P. I., and Geron, C.: Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature), Atmos. Chem. Phys., 6, 3181-3210, doi:10.5194/acp-6- 3181-2006, 20063.\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": "56026", "date": "12 Nov 2019", "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:\nThe article is within the scope of the F1000 Research Open for Science. The 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. The authors are using modern concepts, pertinent equations and numerical comparisons between relevant variables which are clear to understand; besides, the authors detailed the data validation process with tools for spectral signatures.\nAlthough the authors quantify the emissions of natural volatile organic compounds produced by the forest species: Eucalyptus globulus L., Pinus radiata and Alnus acuminata in Riobamba, Ecuador, in the \"Results and Discussions\" the authors need to discuss the limitations of this study.\nSpecific comments (Please make corresponding adjustments):\nIn Table 1, lines: 2, 4 y 5 the terms “High tress” should be replaced by “High trees”.\n\nThe text included in all figures should be improved with more resolution and with uniform font sizes.\n\nOn page 5, “Measurement of volatile organic compounds (COV) and NO2” should be replaced by “Measurement of volatile organic compounds (VOC) and NO2”.\n\nOn page 9, figure 9 “OCOV” should be replaced by “OVOC” or BVOC?\n\nOn page 11, figure 11 “OCOV” should be replaced by “OVOC” or BVOC?\n\nOn page 11, figures 12 “COV” should be replaced by “VOC”.\n\nOn page 12, figures 13 “COV” should be replaced by “VOC”.\n\nOn page 13, “… OCOV (Table 1)” must be confirmed if the authors refer to “other volatile organic compounds OVOC”, or “oxygenated volatile organic compounds OVOC” or BVOC? Please make corresponding adjustments.\n\nRecommendation: The authors should include the following references:\nPréndez, M., Araya, M., Criollo, C., Egas, C., Farías, I., Fuentealba, R., & González, E. (2019). Urban Trees and Their Relationship with Air Pollution by Particulate Matter and Ozone in Santiago, Chile. In Urban Climates in Latin America (pp. 167-206). Springer, Cham.1 González-García, S., Ferro, F. S., Silva, D. A. L., Feijoo, G., Lahr, F. A. R., & Moreira, M. T. (2019). Cross-country comparison on environmental impacts of particleboard production in Brazil and Spain. Resources, Conservation and Recycling, 150, 104434.2\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1012
https://f1000research.com/articles/8-91/v1	23 Jan 19	{ "type": "Case Report", "title": "Case Report: Rare presentation of De Garengeot Hernia", "authors": [ "Tom Crawley-Smith" ], "abstract": "The presentation of an incarcerated appendix within a femoral hernia accounts for 0.5-3.3% of all femoral hernias. It is rarely apparent diagnostically prior to surgery. A 48-year-old female had a delayed presentation with a 3-day history of an irreducible right inguinal swelling. Imaging failed to elucidate an incarcerated appendix, which was found at operation. The patient made a full recovery. The rarity and presentation of this condition is discussed. On literature review it typically is not suspected at operation due to its rarity and the difficulty of interpreting it on examination and imaging.", "keywords": [ "Femoral hernia", "Incarceration", "appendicitis", "De Garengeot Hernia" ], "content": "Introduction\n\nThe presentation of femoral hernia with an incarcerated appendix accounts for 0.5-3.3% of all femoral hernias1; very few cases have been described2. The condition is named after Rene De Garengeot, a French surgeon who first described it in 17313. The condition may be described as the femoral counterpart of the more widely described Amyand hernia, involving appendicitis within the inguinal hernia sac1.\n\n\nPatient information\n\nA 48-year-old woman presented to Dalby Hospital (a small rural facility) with a 3-day history of an irreducible right inguinal swelling, which came on while cycling a mountain bike. A timeline of care is given in Table 1. She had initially not presented as she suspected a muscle strain but presented when the pain became worse. She reported 2–3 previous occurrences of a lump in the same location many years ago, which had self-resolved. Her prior medical history was notable: 13 previous pregnancies with 10 natural deliveries and 3 terminations, LLETZ procedure for cervical cancer, no previous abdominal surgeries. She was an active smoker with a 25 pack-year history. She took no regular medications.\n\n\nClinical examination\n\nThe patient was initially examined by a rural general practitioner who was concerned for incarcerated hernia. He discussed the case with the surgical registrar at Toowoomba hospital and arranged for interhospital transfer. On transfer that evening to Toowoomba Hospital (Toowoomba, Queensland, Australia), a regional facility with 240 beds, the swelling was red and inflamed. The patient was haemodynamically stable, afebrile and was moving her bowels. A right sided, painful swelling could be palpated in the right inguinal region. The registrar examining the patient was suspicious for an incarcerated femoral hernia. In the absence of obstructive symptoms it was suspected that this was incarcerated fat only.\n\n\nInvestigations\n\nIn order to exclude the more serious diagnosis of incarcerated bowel with the hernia and to confirm the diagnosis of femoral hernia, an initial ultrasound was ordered by the treating surgical registrar after discussion with the consultant of the night. In addition a full blood count and electrolytes with liver function tests was ordered. The blood tests were all in the normal range. Meanwhile the ultrasound was reported as an inguinal hernia with suspicion of incarcerated small bowel. The discrepancy of this radiological finding with the clinical findings caused further discusion between the radiology sonographer, consultant and the surgical team; the diagnosis of the type of hernia and its contents mandated the urgency of theatre, approaches and timing. As a result, a CT was ordered to further investigate the anatomy in this case.\n\nThe initial findings of the CT scan were suggestive of an inflamed inguinal hernia with predominant fat contents and probable bowel involvement. There was no radiographic evidence of a small or large bowel obstruction. As this patient’s bowels were still moving, it was felt that this was most likely caused by incarcerated, strangulated fat, rather than bowel.\n\nThe patient was taken to theatre. Because of the above findings, an incision was made over the inguinal ligament, expecting to find an incarcerated inguinal hernia. Instead, on dissection, a femoral hernia was encountered. The sack was opened and necrotic mucosal content was encountered. It was suspected that this was necrotic bowel requiring resection, so the decision was made a low midline laparotomy to ensure safe resection. On opening, a necrotic appendix was found to be incarcerated in the femoral hernia.\n\nThe appendix being reduced, the mesoappendix was clamped, divided and ligated, and the appendix was removed with a purse string suture used to bury residual mucosa. The femoral hernia was repaired primarily with nylon to the conjoin tendon after excision of the sac. A Blake drain was placed and laparotomy wounds were closed with looped Novafil sutures. The patient was placed on Ceftriaxone, 1 g daily and metronidazole 500 mg BD.\n\nThe patient recovered swiftly, and was discharged on day 3 following surgery (Table 1). Her antibiotics were ceased after 24 hours and she was treated with simple analgesia only as required. She was subsequently seen in the outpatients’ clinic at 2 weeks after surgery, and had made a full recovery.\n\n\nDiscussion\n\nThis case was notable for its rarity and the clinical and radiological difficulty anticipating the incarceration of the appendix in the femoral canal. This might have mandated a different approach on surgery than might have been undertaken. Accurate diagnosis of the condition would allow for appropriate choosing of incision, or a laparoscopic approach. On review of the literature, this is typical of this rare condition, however. Excepting one Japanese study4, the diagnosis was typically made serendipitously at surgery. This is not unique, to De Garengeot’s hernia; there can often be confusion between femoral hernia and inguinal hernias, particularly upon clinical examination. Littre’s hernia containing Meckel’s diverticulum and a Richter’s hernia are often also diagnosed on the table rather than in the radiologist’s suite.\n\nThe rarity of this condition contributes to this. De Garengeot’s is an exceedingly rare condition, accounting for 0.5–3.3% of femoral hernias1, which are rare in and of themselves, accounting for only 3–5% of all presentations of hernia. This can make clinical and radiological suspicion all the more challenging.\n\nThe suspicion of a De Garengeot’s hernia earlier on imaging would have dictated either a laparoscopic approach, which may have also been useful to confirm the diagnosis, or a midline mini-laparotomy. Laparoscopic repair has been described as feasible for treatment of this condition; however, its rarity and difficulty of diagnosis prior to operation would make prospective comparison extremely difficult.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nConsent\n\nWritten consent for publication of their clinical details was obtained from the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nVoitk AC, McFarlane JK, Estrada RL: Ruptured appendicitis in femoral hernias: report of two cases and review of the literature. Ann Surg. 1974; 179(1): 24–26. PubMed Abstract \| Free Full Text\n\nTalini C, Oliveira LO, Araújo AC, et al.: De Garengeot hernia: case report and review. Int J Surg Case Rep. 2015; 8C: 35–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGillion JF, Bornet G, Hamrouni A, et al.: Amyand and de Garengeot' hernias. Hernia. 2007; 11(3): 289–290. PubMed Abstract \| Publisher Full Text\n\nEbisawa K, Yamazaki S, Kimura Y, et al.: Acute Appendicitis in an Incarcerated Femoral Hernia: A Case of De Garengeot Hernia. Case Rep Gastroenterol. 2009; 3(3): 313–317. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "45339", "date": "08 Mar 2019", "name": "Zhamak Khurgami", "expertise": [ "Reviewer Expertise Surgery", "gastrointestinal diseases" ], "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 table could be deleted, and the table information explained in the text. The names of all the hospitals are not necessary to mention. Please provide a picture from the CT scan if possible. Please discuss if the patient could be managed without the use of US and CT scans and just explore the incarcerated hernia. Please discuss if, based on available evidence, we could use the mesh in this situation. Please explain which structure was sutured to the conjoint tendon. Many details in the presentation and technique could be deleted.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "4504", "date": "05 Jul 2019", "name": "Tom Crawley-Smith", "role": "Author Response", "response": "Thank you for your feedback.I agree with the comment regarding the table. I originally did supply the time course within the text but was told to include this as a timeline by the journal for submission. I have included it but would be very happy to revert to how it was previously.I have added images from the CT, Ultrasound and intra operative findings which hopefully illuminate the case. Ultimately we found the imaging did not actually impact our decision to take the patient to theatre or help us with deciding on initial incision. In retrospect it could be argued simply proceeding to theatre without imaging would have been an equal and superior outcome in terms of time. I have adjusted my comments to this in the discussion.In terms of our femoral hernia repair after the excision of the incarcerated appendix, we performed a Bassini repair by suturing the conjoin tendon to the inguinal ligament to close the potential space with nylon. Mesh repair was considered but given the resection of the appendix it was felt to be too high a potential risk of infection" } ] } ]	1	https://f1000research.com/articles/8-91
https://f1000research.com/articles/8-312/v1	20 Mar 19	{ "type": "Case Report", "title": "Case Report: Acute pyelonephritis and hearing loss in scrub typhus", "authors": [ "Sivaranjini Venketesan", "Dheeraj Jain", "Stalin Viswanathan", "Murugesan Sivagurunathan Gayathri", "Sivaranjini Venketesan", "Dheeraj Jain", "Murugesan Sivagurunathan Gayathri" ], "abstract": "Acute pyelonephritis is a common renal manifestation in patients with diabetes. A 52-year-old diabetic lady presented with loin pain, dysuria, and fever and urinary incontinence that had begun seven and three days prior to presentation respectively. She was treated with escalating spectra of intravenous antibiotics without improvement. Urine and blood cultures were sterile, while radiological investigations were suggestive of pyelonephritis. Mild hepatic dysfunction prompted consideration of scrub typhus and she improved with empirical doxycycline. Scrub IgM was later confirmed to be positive. In conclusion, local prevalence of systemic infections such as rickettsioses should always be considered in diabetics with fever, even if symptoms and signs otherwise suggest typical diabetes-related infections. We, therefore report a case of acute pyelonephritis caused by scrub typhus which has not been previously described in English medical literature.", "keywords": [ "scrub typhus", "acute pyelonephritis", "urinary tract infection", "hearing loss" ], "content": "Introduction\n\nAmong patients with diabetes mellitus, the urinary tract is the most common site of infection1. Urinary tract infections (UTI) are either related to the upper or lower urinary tract. Acute and chronic pyelonephritis are upper UTIs1. Bacteria (Escherichia coli), viruses (Adenovirus), fungi (Mucor), and mycobacteria (Mycobacterium tuberculosis) commonly cause upper UTIs in diabetes1. Orientia tsutsugamushi (scrub typhus) has never been reported to cause pyelonephritis in English medical literature.\n\n\nCase report\n\nA 52-year-old grandmother of Indian origin, non-compliant to insulin for six months, presented to the Emergency Department of our hospital with fever and rigors, vomiting, headache, bilateral leg pain and myalgia, which had persisted for one week and urinary incontinence for the prior three days. On examination, she was conscious, oriented, toxic, febrile, drowsy, dehydrated with slurred speech, with body-mass index 20.2 kg/m2, tachycardia, orthostatic hypotension, diminished hearing, with right renal angle fullness and tenderness. Initial investigations (Table 1) revealed random sugars 435mg/dL, normal renal functions, ketonuria and glycosuria without pyuria, sinus tachycardia (electrocardiogram), and normal echocardiography. Arterial blood gas showed respiratory alkalosis with metabolic acidosis. Intravenous ceftriaxone 2g OD, intravenous fluids, insulin, acetaminophen 500mg three times a day, multivitamins (B12 1000µg, thiamine 100mg, pyridoxine 100mg, riboflavin 5mg and folate 5mg), pantoprazole 40mg, and domperidone 10mg were commenced for probable acute pyelonephritis. On day 3, piperacillin/tazobactam 4.5g every 8 hours and fluconazole 300mg once a day (OD) were substituted for ceftriaxone 2 g OD; oral amitriptyline 25mg was added to treat the patient’s painful neuropathy.\n\nNormal ranges for each test are provided. ESR - erythrocyte sedimentation rate.\n\nBlood and urine cultures were sterile. Ultrasonogram showed hepatomegaly and bilateral bulky kidneys. She developed diarrhea on day 4. Hence, piperacillin was discontinued after 36hrs even though there appeared to be a partial defervescence; diarrhea subsided after discontinuing piperacillin. Her toxemia and prostration persisted, and she needed assistance to the toilet in view of extreme weakness, but had no focal neurological deficits. On day 5, she was initiated on meropenem 1g every 8 hours and linezolid 600mg every 12 hours for persisting fever (Figure 1A). Liver function tests showed elevated transaminases; hence rickettsioses was suspected and empirical doxycycline 100mg twice a day was initiated on day 6. The next day, Scrub IgM returned positive. Intravenous antibiotics were discontinued and there was no recurrence of fever thereafter.\n\n(A) Fever spikes plotted from day 1 to day 5. (B) Computed tomography scan revealing bilateral renomegaly and mild fat stranding in the right kidney.\n\nAbdominal computed tomography (CT) on the 8th day showed bilateral bulky kidneys with mild perinephric fat stranding (Figure 1B), thus confirming the provisional diagnosis. She also had left-sided proliferative diabetic retinopathy and bilateral sensorineural hearing loss (average of 75dB and 90dB in the right and left ear respectively). She then completed a 7-day course of doxycycline 100mg twice a day and was advised another week’s therapy at home. She had had no fever thereafter. On follow up, liver function test (LFT) had improved (Table 1), as did her hearing (47 dB in the right and 87 dB in the left) which favored our diagnosis of acute scrub typhus.\n\n\nDiscussion\n\nDiabetes mellitus, due to hyperglycemia, ketoacidosis, vascular insufficiency, and impaired neutrophil and monocyte function, makes patients prone to UTIs1. Diagnosis of acute pyelonephritis (APN) clinically is a syndrome of fever, chills, vomiting, and flank pain associated with pyuria, and is often radiologically confirmed2. In a prospective study, only 1/4th patients had a positive urine culture and only 65% had pyuria2, echoing the findings in our patient. In total, 14 among 223 patients had diabetes. Even though our patient did not have pyuria, symptoms/signs in a poorly controlled diabetic led us to a diagnosis of APN. Renal abnormalities in scrub typhus range from simple proteinuria/hematuria to acute kidney injury and occasionally, chronic kidney disease3. Our patient had glycosuria and positive microalbumin (92µg/mg). Mechanisms postulated for renal involvement include rickettsiae-related vasculitis, tubular interstitial proliferation, and tubular necrosis3. APN in scrub typhus has been reported only once, in Chinese medical literature in a 56-year-old Chinese lady who had urgency, flank pain and an eschar4.\n\nDiabetes is a risk factor for scrub typhus-induced acute kidney injury. Since leukocytosis reduced with ceftriaxone without adequate fever response, we presumed poor control of bacterial/fungal infection and treated her with fluconazole and piperacillin/tazobactum. Since LFT was not performed prior to day 4 due to technical reasons, rickettsioses were not suspected. Though our locality is a high prevalence area for scrub typhus, focal renal signs and symptoms led us to think otherwise5. We also erred in contributing her hearing impairment to be the result of her toxemia and poor health. Pure tone audiometry was done 48 hours after doxycycline when the patient became self-ambulatory. Improvement of her hearing loss, albeit partial, two weeks after discharge suggests that scrub typhus could have also contributed to her hearing impairment6. Abdominal CT was also done after doxycycline therapy-whether findings are milder than expected is also debatable. Hypoalbuminemia and rapidly falling hemoglobin over seven days without overt blood or volume loss, could be attributed to hemoconcentration following scrub typhus-related capillary leak syndrome that was observed at initial presentation, and reverted to premorbid levels after fluid supplementation and antibiotics. In retrospect, fever, absence of pyuria, sterile urine, capillary leak syndrome, and primary respiratory alkalosis in a patient with high sugars and ketonuria should have made us think of an alternative etiological diagnosis. Deranged transaminases nudged us in the right direction.\n\nUTIs in diabetes are common, but scrub typhus as a probable cause of UTI/pyelonephritis has hitherto been unreported in English medical literature. Atypical organisms causing pyelonephritis should be considered in patients with multisystem involvement and in those with a UTI but without pyuria. Furthermore, local prevalence of systemic infections such as rickettsioses should always be considered in diabetics with fever, even if symptoms and signs otherwise suggest typical diabetes-related infections.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images were 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": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBalachandar MS, Pavkoviç P, Metelko Ž: Kidney infections in diabetes mellitus. Diabetol Croat. 2002; 31(2): 85–103. Reference Source\n\nRollino C, Beltrame G, Ferro M, et al.: Acute pyelonephritis in adults: a case series of 223 patients. Nephrol Dial Transplant. 2012; 27(9): 3488–93. PubMed Abstract \| Publisher Full Text\n\nHwang K, Jang HN, Lee TW, et al.: Incidence, risk factors and clinical outcomes of acute kidney injury associated with scrub typhus: a retrospective study of 510 consecutive patients in South Korea (2001-2013). BMJ Open. 2017; 7(3): e013882. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShu LH, Xu Y: [One case of scrub typhus patient with clinical manifestation of acute pyelonephritis]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2013; 31(4): 329. PubMed Abstract\n\nVivekanandan M, Mani A, Priya YS, et al.: Outbreak of scrub typhus in Pondicherry. J Assoc Physicians India. 2010; 58: 24–8. PubMed Abstract\n\nPremaratna R, Chandrasena TG, Dassayake AS, et al.: Acute hearing loss due to scrub typhus: a forgotten complication of a reemerging disease. Clin Infect Dis. 2006; 42(1): e6–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "45955", "date": "03 Apr 2019", "name": "Dharshan Rangaswamy", "expertise": [ "Reviewer Expertise Clinical Nephrology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a rare reported manifestation of Scrub Typhus namely acute pyelonephritis and hearing loss which improved with treatment. Acute pyelonephritis has been reported once as a case report from China and hearing loss in another six cases. The case is well described and convincing in that clinical features favor acute pyelonephritis. However since authors are describing a manifestation for the first time a more detailed account would have been better. For example, in view of negative cultures, treatment received prior to entry could have been listed. The authors base their conclusion on a chance sending of scrub IgM which came positive whereas learning points which serve as pointers to the diagnosis may be mentioned in discussion. The presence of liver dysfunction, anemia and fever would have merited investigation with other common tropical infections such as malaria, which can also involve the kidneys.\nUnusual features in this description are absence of eschar, thrombocytopenia for scrub typhus and absence of pyuria. The absence of pyuria in 1/3rd cases as observed in a previous prospective study, had only 6% of diabetics in their cohort and the diagnosis of acute pyelonephritis was mainly based on clinical criteria1. The sensitivity and specificity for diagnosis of acute pyelonephritis on urine investigations are: urinalysis (>10 WBCs/high power field), 58%–82% and 65%–86%; positive leukocyte esterase, 74%–96% and 94%–98%; nitrite, 35%–85% and 92%–100%; and leukocyte esterase plus nitrite tests, 75%–84% and 82%–89% respectively2,3,4,5. The sensitivity and specificity for diagnosis for acute pyelonephritis based on perinephric fat stranding was 72% and 60%. It is affected by age, sex and renal function and may not be a particularly useful tool for the diagnosis of acute pyelonephritis6. Bilateral enlarged kidneys, renal angle tenderness with associated vomiting are also observed in patients with infection or drug related acute interstitial nephritis. However, the absence of pyuria points against the diagnosis.\nCertainly, scrub typhus can cause inflammation of organs due to endothelial infection, vasculitis and increases endothelial permeability. This report highlights the importance of keeping a high index of suspicion for this easily treatable disease especially in endemic areas. The article can be accepted as approved with caveats stated above.\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": "4706", "date": "05 Jul 2019", "name": "Stalin Viswanathan", "role": "Author Response", "response": "Dear Sir,The lady did not receive any prior treatment. The details have been included.It was not a chance sending of Scrub IgM. That facility is not available at our institution. Since LFT was done only on day 4, we could send a sample for Scrub IgM to a private lab only on day5.Malarial parasites were not visualized on peripheral smear. Moreover, the LFT dysfunction was without hyperbilirubinemia.All these details were not mentioned since this manuscript was submitted to another journal previously with a word count of 1000, and such information fell prey to the editing keys.Unusual was the absence of eschar, but it is well known in Indian populations that the incidence of eschar is varying between 4-46% (Vivekanandan et al, 2010).Thrombocytopenia was expected in a case of scrub typhus, and such a finding would have enabled us to pursue a diagnosis of scrub typhus earlier, even before the LFT." } ] }, { "id": "48908", "date": "28 May 2019", "name": "Tri Wangrangsimakul", "expertise": [ "Reviewer Expertise Infectious diseases", "microbiology", "tropical medicine", "rickettsial diseases." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a case of a 52 year old diabetic lady with complications, presenting with pyelonephritis. Cultures were negative and she was treated with antibiotics that were regularly escalated. She was also found to have bilateral sensorineural deafness on audiometry. Elevated liver enzymes prompted testing for scrub typhus and the IgM came back positive, leading to initiation of doxycycline and defervescence within hours.\nFor any case report, the diagnosis must be as robust as possible and an effort made to rule out other possible diagnoses. I agree with the other review that malaria should be ruled out. Other diagnoses to be excluded include leptospirosis, biliary infection or pancreatitis. Other background information would be useful e.g. antibiotics used pre-admission, occupation, freshwater contact, rodent contact, agricultural work, insect bites, alcohol intake and past history of scrub typhus.\nThe basis for the diagnosis of scrub typhus used in this report is limited. A \"'Scrub IgM positive\" does not inform the reader of what serological test or method was used, where the test was performed, what diagnostic cut-off was used to determine a positive result and whether this cut-off has been validated for the population which the patient belongs to. I suspect the test used was the InBios Scrub Typhus IgM ELISA, in which case the OD value and how the diagnostic cut-off was reached should be stated so the reader can form their own opinion on the strength of diagnosis.\nThe diagnosis of scrub typhus is not straightforward 1. Antibody-detection tests (IFA, ELISAs) have inherent weaknesses, such as the presence of circulating antibodies in individuals within an endemic area where repeated exposure occurs, leading to false positive results. Acute and convalescent samples can give a clearer picture and a rise in antibody titre to >= 4 times is usually part of the diagnostic criteria. Is a convalescent blood sample available to compare IgM serology results? Culture and PCR will be more specific and will greatly improve the certainty of diagnosis but may not be available.\nScrub typhus can cause AKI, glomerulonephritis and nephrotic syndrome so pyelonephritis is possible 2,3. However, the diagnostic case for scrub typhus must be more robust than currently presented. Additionally, one could argue that the general trend was that she was improving when doxycycline was initiated on day 6 as observed on the fever chart, which would go against severe scrub typhus with a renal complication.\nTwo main lessons can be drawn from this report. Firstly, in regions where scrub typhus is endemic and accurate diagnostic assays are unavailable, empirical treatment with doxycycline should be considered. Secondly, antibody-detection assays for scrub typhus have inherent weaknesses and until paired antigen/IgM tests are available, acute and convalescent samples are recommended for diagnostic confirmation. In this report, the evidence for scrub typhus infection is currently limited and the conclusion that this diagnosis is established should be delayed until further diagnostic information is available.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "4707", "date": "05 Jul 2019", "name": "Stalin Viswanathan", "role": "Author Response", "response": "Dear Sir,Malarial parasites were not visualized on the peripheral smear on at least 2 occasions. Nevertheless, there was no hepatosplenomegaly, discolored urine, jaundice, hyperbilirubinemia and thrombocytopenia to suggest malaria.The same goes for biliary infection. The lady did not have pain suggestive of pancreatitis and computed tomography of pancreas was normal. We did not perform lipase.Leptospirosis was considered even without her exposure to rain, water, or rodents but the thought did not persist in the absence of renal failure and thrombocytopenia and lack of response to ceftriaxone in the first three days.Background information regarding her employment and others have been added to the manuscript.The diagnostic kit and the cutoff values have been mentioned in the manuscript. Convalescence titers could not be performed due to the cost of performing it in an outside lab.We would not generally agree that she was improving till day 5 even without doxycycline since the toxemia persisted that made us initiate meropenem and linezolid. Her symptoms did not recur on stopping these drugs and initiating doxycycline.Culture and PCR were not available. This has been added to the text." } ] } ]	1	https://f1000research.com/articles/8-312
https://f1000research.com/articles/8-1006/v1	04 Jul 19	{ "type": "Software Tool Article", "title": "Large-scale sequence comparisons with sourmash", "authors": [ "N. Tessa Pierce", "Luiz Irber", "Taylor Reiter", "Phillip Brooks", "C. Titus Brown", "N. Tessa Pierce", "Luiz Irber", "Taylor Reiter", "Phillip Brooks" ], "abstract": "The sourmash software package uses MinHash-based sketching to create “signatures”, compressed representations of DNA, RNA, and protein sequences, that can be stored, searched, explored, and taxonomically annotated. sourmash signatures can be used to estimate sequence similarity between very large data sets quickly and in low memory, and can be used to search large databases of genomes for matches to query genomes and metagenomes. sourmash is implemented in C++, Rust, and Python, and is freely available under the BSD license at http://github.com/dib-lab/sourmash.", "keywords": [ "sequence analysis", "MinHash", "k-mer", "sourmash", "bioinformatics" ], "content": "Introduction\n\nBioinformatic analyses rely on sequence comparison for many applications, including variant analysis, taxonomic classification and functional annotation. As the Sequence Read Archive now contains over 20 Petabases of data1, there is great need for methods to quickly and efficiently conduct similarity searches on a massive scale. MinHash techniques2 utilize random sampling of k-mer content to generate small subsets known as \"sketches\" such that Jaccard similarity (the intersection over the union) of two sequence data sets remains approximately equal to their true Jaccard similarity2,3. The many-fold size reductions gained via MinHash opens the door to extremely large scale searches.\n\nWhile the initial k-mer MinHash implementation focused on enabling Jaccard similarity comparisons3, it has since been modified and extended to enable k-mer abundance comparisons4, decrease runtime and memory requirements5, and work on streaming input data6. Furthermore, as Jaccard similarity is impacted by the relative size of the sets being compared, containment searches2,7,8 have been developed to enable detection of a small set within a larger set, such as a genome within a metagenome.\n\nHere we present version 2.0 of sourmash9, a Python library for building and utilizing MinHash sketches of DNA, RNA, and protein data. sourmash incorporates and extends standard MinHash techniques for sequencing data, with a particular focus towards enabling efficient containment queries using large databases. This is accomplished with two modifications: (1) building sketches via a modulo approach2, and (2) implementing a modified Sequence Bloom Tree10 to enable both similarity and containment searches. In most cases, these features enable sourmash database comparisons in sub-linear time.\n\nStandard genomic MinHash techniques, first implemented in Ondov BD et al.3, retain the minimum n k-mer hashes as a representative subset. sourmash extends these methods by incorporating a user-defined \"scaled\" factor to build sourmash sketches via a modulo approach, rather than the standard bottom-hash approach2. Sketches built with this approach retain the same fraction, rather than number, of k-mer hashes, compressing both large and small datasets at the same rate.\n\nThis enables comparisons between datasets of disparate sizes but can sacrifice some of the memory and storage benefits of standard MinHash techniques, as the signature size scales with the number of unique k-mers rather than remaining fixed8. In sourmash, use of the \"scaled\" factor enables user modification of the trade-off between search precision and sketch size, with the caveat that searches and comparisons can only be conducted using signatures generated with identical \"scaled\" values (downsampled at the same rate).\n\nTo enable large-scale database searches using these signatures, sourmash implements a modified Sequence Bloom Tree (SBT), the SBT-MinHash (SBTMH), that allows both similarity (sourmash search) and containment (sourmash gather) searches for taxonomic exploration and classification. Notably, Jaccard similarity searches using this modified SBT require storage of the cardinality of the smallest MinHash below each node in order to properly bound similarity. sourmash also implements a second database format, \"LCA\", for in-memory search when sufficient RAM is available or database size is tractable. The LCA format can be leveraged to return additional information, such as taxonomic lineage.\n\nIn addition to these modifications, sourmash implements k-mer abundance tracking4 within signatures to allow abundance comparisons across datasets and facilitate metagenome, metatranscriptome, and transcriptome analyses, and is compatible with streaming approaches. The sourmash library is implemented in C++, Rust11, and Python, and can be accessed both via command line and Python API. The code is available under the BSD license at http://github.com/dib-lab/sourmash.\n\n\nImplementation\n\nsourmash provides a user-friendly, extensible platform for MinHash signature generation and manipulation for DNA, RNA, and protein data. Sourmash is designed to facilitate containment queries for taxonomic exploration and identification while maintaining all functionality available via standard genomic MinHash techniques.\n\nsourmash modifies standard genomic MinHash techniques in two ways. First, sourmash scales the number of retained hashes to better represent and compare datasets of varying size and complexity. Second, sourmash optionally tracks the abundance of each retained hash, to better represent data of metagenomic and transcriptomic origin and allow abundance comparisons.\n\nScaling. sourmash implements a method inspired by modulo sketches2 to dynamically scale hash subset retention size (n). When using scaled signatures, users provide a scaling factor (s) that divides the hash space into s equal bands, retaining hashes within the minimum band as the sketch. These scaled signatures can be converted to standard bottom-hash signatures, if the subset retention size n is equal to or smaller than the number of hashes in the scaled signature. sourmash provides a signature utility, downsample, to convert sketches when possible. Finally, to maintain compatibility with sketches generated by other programs such as Mash3, sourmash generates standard bottom-hash MinHash sketches if users specify the hash subset size n rather than scaling factor.\n\nStreaming compatibility. Scaled signature generation is streaming compatible and provides some advantages over streaming calculation using standard MinHash. As data streams in, standard MinHash replaces hashes based on the minimum hash value to maintain a fixed number of hashes in the signature. In contrast, no hash is ever removed from a scaled signature as more data is received. As a result, for searches of a database using streamed-in data, all prior matches remain valid (although their significance may change as more data is received). This allows us to place algorithmic guarantees on containment searches using streaming data.\n\nAbundance tracking. sourmash extends MinHash functionality by implementing abundance tracking of k-mers. k-mer counts are incremented after hashing as each k-mer is added to the hash table. sourmash tracks abundance for k-mers in the minimum band and stores this information in the signature. These values accompany the hashes in downstream comparison processes, making signatures better representations of repetitive sequences of metagenomic and transcriptomic origin.\n\nSignatures. MinHash sketches associated with a single sequence file are stored together in a “signature” file, which forms the basis of all sourmash comparisons. Signatures may include sketches generated with different k sizes or molecule type (nucleotide or protein) and are stored in JSON format to maintain human readability and ensure proper interoperability.\n\nSignatures can only be compared against signatures and databases made from the same parameters (k size(s), scaled value, nucleotide or protein level). If signatures differ in their scaled value or size(n), the larger signatures can be downsampled to become comparable with smaller signatures using the signature utilities, below. sourmash also provides 6-frame nucleotide translation to generate protein signatures from nucleotide input if desired.\n\nSignature utilities. sourmash provides a number of utilities to facilitate set operations between signatures (merge, intersect, extract, downsample, flatten, subtract, overlap), and handling (describe, rename, import, export) of sourmash signatures. These can be accessed via the sourmash signature subcommand.\n\nsourmash implements a modified Sequence Bloom Tree (SBT10), the SBT-MinHash (SBTMH), which can efficiently capture large volumes of MinHashes (e.g., all microbes in GenBank) and support multiple search regimes that improve on run time of linear searches.\n\nImplementation. The SBTMH is a n-ary tree (binary by default), where leaf nodes are MinHash signatures and internal nodes are Bloom Filters. Each Bloom Filter contains all the values from its children, so the root node contains all the values from all signatures. SBTMH is designed to be extensible such that signatures can be subsequently added without the need for full regeneration. Adding a new signature to SBTMH causes parent nodes up to the root to be updated, but other nodes are not affected.\n\nSBTMH trees can be combined if desired: In the simplest case, adding a new root and updating it with the content of the previous roots is sufficient, and this preserves all node information without changes. As an example, separate indices can be created for each RefSeq subdivision (bacteria, archaea, fungi, etc) and be combined depending on the application (such as an analysis for bacteria + archaea, but not fungi). In practice, this is most useful for updating the SBTMH, as both search and gather support search over multiple databases without the need for rebuilding a single large database.\n\nSearching SBTMH. Similarity searches start at the root of the SBTMH, and check for query elements present in each internal node. If the similarity does not reach the threshold, the subtree under that node does not need to be searched. If a leaf is reached, it is returned as a match to the query signature. In order to enable similarity (in addition to containment) searches using this modified SBTMH, nodes store the cardinality of the smallest signature below each node in order to properly bound similarity. The full SBTMH does not need to be imported to RAM during searches, making this method best for rapid searching with minimal memory requirements. However, if sufficient RAM is available, searches of databases (or many signatures) may be completed in memory via an alternate database format (discussed below).\n\nSBTMH utilities. sourmash provides several utilities for construction, use, and handling of SBTMH databases. These include sbt index to index a collection of signatures as an SBTMH for fast searching, sbt append to add signatures, and sbt combine to join two SBTMH databases.\n\nsourmash implements an alternate database format, LCA, to support in-memory queries. This implementation utilizes two named lists to store MinHashed databases: the first containing MinHashes, and the second containing taxonomic information, with both lists named by sample name. This structure facilitates direct look-up of MinHashes, and thus can be leveraged to return additional information, such as taxonomic lineage. The LCA database structure can be prepared using the sourmash lca index command.\n\n\nAssessing sequence similarity\n\nFor sequence comparison, sourmash compare reimplements Jaccard sequence similarity comparison to enable comparison between scaled MinHashes. When abundance tracking of k-mers is enabled, compare instead calculates the cosine similarity, although we recommend using more accurate approaches for detailed comparisons6.\n\nIn addition to conducting pairwise comparisons, two types of database searches are implemented: breadth-first similarity searches (sourmash search) and best-first containment searches (sourmash gather), which support different biological queries. These searches can be conducted using either database format.\n\nSimilarity queries. Breadth-first sourmash search can be used to obtain all MinHashes in the SBTMH that are present in the query signature (above a specified threshold). This style of search is streaming-compatible, as the query MinHash can be augmented as the search is occurring.\n\nContainment queries Best-first sourmash gather implements a greedy algorithm where the SBTMH is descended on a linear path through a set of internal nodes until the highest containment leaf is reached. The hashes in this leaf are then subtracted from the query MinHash and the process is repeated until the threshold minimum is reached. sourmash post-processes similarity statistics after the search such that it reports percent identity and unique identity for each match.\n\nTaxonomy-resolved searches sourmash can conduct taxonomy-resolved searches uses the “least common ancestor” approach (as in Kraken12), to identify k-mers in a query. From this it can either find a consensus taxonomy between all the k-mers (sourmash classify) or it can summarize the mixture of k-mers present in one or more signatures (sourmash summarize).\n\nsourmash is a tool for building and utilizing MinHash signatures of DNA, RNA, and protein sequences. A straightforward workflow consists of generating a signature using sourmash compute, and comparing it against other signatures or databases of signatures via sourmash compare, search, gather, lca search, or lca gather. sourmash has no particular memory requirements, but does need to hold the largest single sequence in memory while generating a signature. For example, computing a signature from a 100Mb human microbiome sample requires 30MB of RAM, and searching it against a sourmash Genbank signature database takes 1–6 minutes and requires 2–6 GB of RAM, depending on the search type. \"LCA\" databases are smaller on disk but require more memory to be searched.\n\nBelow we provide several use cases to demonstrate the utility of sourmash for sequence comparisons, starting with signature generation and proceeding into signature comparisons, tetranucleotide frequency clustering analysis, and taxonomic classification. We primarily demonstrate nucleotide-level applications in this paper; protein-level analyses will be explored further in future work. Additional information and tutorials are available at https://sourmash.readthedocs.io.\n\n\nUse cases\n\nsourmash is available for both Linux and OSX, and runs under either Python 2.7.x or Python 3.5+. To install sourmash, we recommend using conda. For these examples, we used sourmash v.2.0.1, installed with conda v 4.6.14.\n\n\n\nAlternate installation instructions are available at sourmash.readthedocs.io.\n\nAll sourmash comparisons work on signatures, compressed representations of biological sequencing data. To create a signature from sequences with abundance tracking:\n\n\n\nBecause a signature can contain multiple MinHashes, multiple k-sizes can be specified per a sequence. Only one scaled size can be used.\n\nBy default, the name of the file becomes the name of the signature. To name the signature from the first line of the sequencing file, use −−name−from−first. Although the −−track−abundance flag is optional, since downstream comparison methods contain the flag −−ignore−abundance to ignore them, we recommend calculating all signatures with abundance tracking.\n\nTo create a signature from protein sequences:\n\n\n\nSignatures can also be made directly from reads. Depending on the downstream use cases, we recommend different preparation methods. When the user aims to compare the signature to other signatures, we recommend k-mer trimming the reads before computing the signature. Because compare does an all-by-all comparison of signatures, errors in the reads will falsely deflate the similarity metric. We recommend trimming RNA-seq or metagenome reads with trim−low−abund.py in the khmer package13, a dependency of sourmash.\n\n\n\nWhen using methods that compare a signature against a database such as gather or search, k-mer trimming need not be used. These methods use exact matching of hashes in the signature to those in the databases. k-mer trimming could increase false negatives, but results on k-mer trimmed data will more accurately represent the proportions of content in the data.\n\n\n\nSignatures calculated with abundance tracking enable rapid comparison of sequences where k-mer frequency is variable, and can be leveraged for quality control and summarization methods. For example, principle component analysis (PCA) and multidimensional scaling (MDS) are standard quality control and summarization methods for count data generated during RNA-seq analysis14. sourmash can be used to build this MDS plot in a reference-free (or assembly-free) manner, using k-mer abundances of the input reads. We also find this useful for comparing other types of RNA sequencing samples (mRNA, ribo-depleted, 3’ tag-seq, metatranscriptomes, and transcriptomes).\n\nHere, we use a set of four Saccharomyces cerevisiae RNA-seq samples: replicate wild-type samples and replicate mutant (SNF2) samples15. To use sourmash to build an MDS plot, we first trim the data to remove low abundance k-mers via khmer13. We demonstrate the streaming capability of sourmash by downloading, k-mer trimming, and calculating a signature with one command. This allows the user to generate signatures without needing to store large files locally.\n\n\n\nThe signature will be named from the input filename, in this case −. We can change the name to reflect its contents using the signature rename function.\n\n\n\nWe can also check that the name has been changed.\n\n\n\nUsing signatures from four samples, we can compare the files with the compare function. Here we download signatures calculated and renamed using the above commands. We output the comparison matrix as a csv for downstream use in other platforms.\n\n\n\nWe then import the compare similarity matrix into R (v3.4.1) to produce an MDS plot with wild-type samples (ERR459011, ERR459102) in yellow and mutant samples (ERR458584, ERR458829) in blue.\n\n\n\nFor comparison, we also produced an MDS plot using a more traditional approach, utilizing Salmon (v0.11.3)16 to quantify abundance relative to an S. cerevisiae reference, and edgeR (v3.22.5)17 to build an MDS plot (Figure 1; code available online at https://osf.io/97rt4/).\n\nWe can also use sourmash with abundance tracking for tetranucleotide frequency clustering. Tetranucleotide usage is species-specific, with strongest conservation in DNA coding regions18. This is often used in metagenomics as one method to “bin” assembled contiguous sequences together that are from the same species19. Recently, tetranucleotide frequency clustering using sourmash was used to detect microbial contamination in the domesticated olive genome20. Here we reimplement this approach using 100 of the 11,038 scaffolds in the draft genome. We calculate the signature using a k-mer size of 4, use all 4-mers, and track abundance. Then we use sourmash compare to calculate the similarity between each scaffold. (The −−singleton flag calculates a signature for each sequence in the fasta file.)\n\nWild-type S. cerevisiae samples (ERR459011, ERR459102) are in yellow and mutant samples (ERR458584, ERR458829) in blue.\n\n\n\nAlthough sourmash compare supports export to a csv file, sourmash also has a built in visualization function, plot. We will use this to visualize scaffold similarity.\n\n\n\nIn Figure 2, we see that there is high similarity between 98 of the scaffolds, but that Oe6_s01156 and Oe6_s01003 are outliers with tetranucleotide frequency similarity around 40% to olive scaffolds. These two scaffolds are contaminants20.\n\nTwo scaffolds are outliers when using tetranucleotide frequency to calculate similarity (highlighted in green on the dendrogram).\n\nMinHash comparisons are useful for outlier detection. Below we compare 50 genomes that contain the word \"Escherichia coli.\" We have pre-calculated the signatures for each of these genomes. We then use the plot function to visualize our comparison.\n\n\n\nWe see that the minimum similarity in the matrix is 0%. If all 50 signatures were from the same species, we would expect to observe higher minimum similarity at a k-mer size of 31. When we look closely, we see one signature has 0% similarity with all other signatures because it is a phage (Figure 3).\n\nOne signature is an outlier (highlighted in blue on the dendrogram).\n\nThe search and gather functions allow the user to classify the contents of a signature by comparing it to a database of signatures. Prepared databases for microbial genomes in RefSeq and GenBank are available at https://sourmash.readthedocs.io/en/latest/databases.html. However, it is also simple to create a custom database with signatures.\n\nBelow we make a database that contains 50 Escherichia coli genomes.\n\n\n\nThis database can be queried with search and gather using any signature calculated with a k-size of 31.\n\nFor example, below we download a small set of k-mer trimmed Escherichia coli reads and generate a signature with k=31.\n\n\n\nThen, we search the 50-genome database created above.\n\n\n\n\n\nBreadth-first sourmash search finds all matches in the SBTMH that are present in the query signature (above a specified threshold).\n\nNow try the same search using sourmash gather.\n\n\n\n\n\nBest-first sourmash gather finds the best match first, e.g. here the first E. coli genome has an 83% match to 65.4% of our query signature. The hashes that matched (65.4% of the query) are then subtracted, and the database is queried with the remaining hashes (34.6% of original query). This process is repeated until the threshold is reached. sourmash post-processes similarity statistics after the search such that it reports percent identity and unique identity for each match.\n\nsourmash gather is also useful for rapid metagenome decomposition. Below we calculate a signature of a metagenome using raw reads, and then use gather to perform a best-first search against all microbial genomes in Genbank. This approach was recently used to classify unknown genomes in a \"mock\" metagenome21. The mock community was made to contain 64 genomes, but additional genomic material was inadvertently added prior to sequencing. Below we will use gather to investigate the content in the mock metagenome that did not map to the 64 reference genomes. For details on how this signature was created, please see Awad et al.22. Note that the GenBank database is approximately 7.8 Gb compressed, and 50 Gb decompressed. Searches of the current Gen-Bank database run fastest if allowed to use 11 Gb of RAM.\n\n\n\nThe output to the terminal begins:\n\n\n\n\n\nWe see that 20.1% of k-mers match 82 genomes in GenBank. The majority of matches are to genomes present in the mock community. However, some species like Proteiniclasticum ruminis were not members of the mock community. These results also highlight how sourmash gather behaves with inexact matches such as strain variants. For example, we see two matches between P. ruminis strains among all matches. This likely indicates that a P. ruminis strain that has not been sequenced before is in our sample, and that it shares more k-mers of size 31 in common with one strain than the other. (See Brown CT et al.23 for further analysis of this strain.)\n\nsourmash gather and search also support custom databases. Using a custom database with sourmash gather, we can identify the dominant contamination in the domesticated olive genome20. Below, we will use a database containing all fungal genomes in NCBI. We will then use the streaming compatibility of sourmash to download and calculate the signature. Lastly, we will search the olive genome against the fungal genomes using gather.\n\n\n\nUsing gather, we see two matches both within the genus Aureobasidium. This is the dominant contaminant within the genome20.\n\n\n\n\nConclusions\n\nThe sourmash package provides a collection of tools to conduct sequence comparisons and taxonomic classification, and makes comparison against large-scale databases such as GenBank and SRA tractable on laptops. sourmash signatures are small and irreversible, which means they can be used to facilitate pre-publication data sharing that may help improve classification databases and facilitate comparisons among similar datasets.\n\n\nData availability\n\nOpen Science Framework: sourmash-use-cases. https://doi.org/10.17605/OSF.IO/KESH2\n\nThis project contains the following underlying data:\n\ndata-files\n\n– ecoli-reads.khmer.fq.gz (Small set of k-mer trimmed Escherichia coli reads)\n\n– ERR458584.fq.gz (Saccharomyces cerevisiae SRA Record ERR458584 SNF2 mutant,15)\n\n– Oe6.scaffolds.fa.gz (domesticated olive (Olea europaea) genome20)\n\n– Oe6.scaffolds.sub.fa.gz (Subsampled set of scaffolds from the domesticated olive (Olea europaea) genome20)\n\n– yeast_ktrimmed.tar (kmer-trimmed Saccharomyces cerevisiae reads15)\n\nindex-files\n\n– escherichia-sigs.tar.gz (Sourmash signatures of 50 randomly selected Escherichia coli genomes)\n\n– fungi-genomic.tar.gz (Sourmash signature database of all fungal genomes in NCBI as of 12/2018)\n\nsignature-files\n\n– GCF_000005845.2_ASM584v2_genomic.fna.gz (Escherichia coli genome str. K-12 substr. MG1655)\n\n– GCF_000146045.2_R64_genomic.fna.gz (Saccharomyces cerevisiae S288C genome)\n\n– GCF_000146045.2_R64_protein.faa.gz (Saccharomyces cerevisiae S288C protein sequence)\n\nOpen Science Framework: sourmash-use-cases. https://doi.org/10.17605/OSF.IO/KESH2\n\nThis project contains the following extended data:\n\nyeast-mds.txt (Code to generate MDS plots via Salmon and edgeR)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSource code available from: https://github.com/ dib-lab/sourmash/\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.3240653\n\nLicence: 3-Clause BSD License", "appendix": "Grant information\n\nThis work is funded in part by the Gordon and Betty Moore Foundation’s Data-Driven Discovery Initiative [GBMF4551 to CTB]. NTP was supported by a National Science Foundation Postdoctoral Fellowship in Biology [1711984].\n\n\nReferences\n\nSequence read archive overview. 2018. Reference Source\n\nBroder AZ: On the resemblance and containment of documents. In Compression and complexity of sequences 1997. proceedings. IEEE. 1997; 21–29. Reference Source\n\nOndov BD, Treangen TJ, Melsted P, et al.: Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016; 17(1): 132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBovee R, Greenfield N: Finch: a tool adding dynamic abundance filtering to genomic minhashing. 2018; 3(22): 505. Publisher Full Text\n\nZhao XF: BinDash, software for fast genome distance estimation on a typical personal laptop. Bioinformatics. 2019; 35(4): 671–673. PubMed Abstract \| Publisher Full Text\n\nRowe WP, Carrieri AP, Alcon-Giner C, et al.: Streaming histogram sketching for rapid microbiome analytics. Microbiome. 2019; 7(1): 40. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrusoe MR, Alameldin HF, Awad S, et al.: The khmer software package: enabling efficient nucleotide sequence analysis [version 1; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2015; 4: 900. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConesa A, Madrigal P, Tarazona S, et al.: A survey of best practices for RNA-seq data analysis. Genome Biol. 2016; 17(1): 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchurch NJ, Schofield P, Gierliński M, et al.: How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? RNA. 2016; 22(6): 839–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatro R, Duggal G, Love MI, et al.: Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14(4): 417–419. 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\nPride DT, Meinersmann RJ, Wassenaar TM, et al.: Evolutionary implications of microbial genome tetranucleotide frequency biases. Genome Res. 2003; 13(2): 145–158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbertsen M, Hugenholtz P, Skarshewski A, et al.: Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat Biotechnol. 2013; 31(6): 533–538. PubMed Abstract \| Publisher Full Text\n\nReiter T, Brown CT: Microbial contamination in the genome of the domesticated olive. 2018. Publisher Full Text\n\nShakya M, Quince C, Campbell JH, et al.: Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities. Environ Microbiol. 2013; 15(6): 1882–1899. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAwad S, Irber L, Brown CT: Evaluating metagenome assembly on a simple defined community with many strain variants. 2017. Publisher Full Text\n\nBrown CT, Moritz D, O’brien M, et al.: Exploring neighborhoods in large metagenome assembly graphs reveals hidden sequence diversity. BioRxiv. 2019; 462788. Publisher Full Text" }	[ { "id": "52588", "date": "27 Aug 2019", "name": "Brad Solomon", "expertise": [ "Reviewer Expertise Computational Biology", "Algorithms and Data Structures", "Genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents an improved software library, sourmash 2.0, for the efficient construction and analysis of MinHash sketches of genomic and proteomic sequence data. The primary innovations of sourmash 2.0 are the novel applications and modified implementations of several existing sketching and indexing methods. In particular, the two main advancements are (1) a ‘modulo approach’ to sketch construction and the development of a modified Sequence Bloom Tree (SBT) index over MinHash sketches. In addition, unlike conventional MinHash methods, sourmash 2.0 can also track the abundance of retained hash elements, allowing some degree of abundance comparison and abundance estimation of genomic datasets.\n\nMultiple use cases for sourmash 2.0 are provided including sketch construction, similarity comparisons, containment querying, classification, and metagenome decomposition. The manuscript is very thorough in describing sketch construction with example run commands for full-length genomes, proteomes, as well as raw reads. The ease of use is further demonstrated with a single line command that, while involving multiple pipes, is capable of constructing a signature through a download data stream. Similarly, several uses of sketch comparisons are demonstrated including visualization and outlier detection. Lastly the use of the SBT MinHash (SBTMH) is demonstrated for classification or containment of arbitrary sequence queries. The download instructions and example run commands are fully functional and the input datasets are reasonably sized for toy examples (most on the order of <100 MB).\nExcluding the description of the modulo approach of sketch construction, the manuscript itself is technically sound. The topic is high impact -- sketching approaches are increasingly popular solutions to a multitude of research topics in computational biology. Many of these potential use cases are demonstrated successfully in the manuscript. That said, there are numerous existing solutions to each of the use cases presented in the manuscript and little to no attempt was made to provide benchmarking information or to demonstrate the improvements sourmash 2.0 has over its competitors. While sourmash 2.0 has an ease of use that will undoubtedly facilitate use, it does not adequately demonstrate an improved capacity for analysis over these other tools. This is primarily a suggestion for improvement as, based on the F1000 Research guidelines, the manuscript is correct and valid in its current state.\nMajor Comments:\nThe ‘modulo approach’ for sketch construction, despite being one of the main innovations of the method, is particularly unclear in the manuscript. The cited literature (Broder 1997) describes an approach that sub-samples hash values based on a modulo factor to address the inherent weakness of a Minhash in a mixture of several distinct components. However the description of the sourmash implementation instead describes splitting the hash space into ‘equal bands’ and selecting only the minimum band. As the existing modulo approach has no guarantees on equal-sized (or even equal-fraction as the manuscript claims elsewhere) sub-sampling, this appears to be a novel and significant contribution to the field. However there are no details that explain (1) how the hash space is divided, (2) how the minimum band is selected, and (3) how downsampling is performed.\n\nSourmash 2.0 is motivated by “a particular focus towards enabling efficient containment queries using large databases”. However the manuscript does not include any true comparisons about sourmash’s performance against existing tools, alternative approaches, or benchmarking information for even conventionally sized datasets. This greatly limits the potential impact of sourmash given there are many competing sketch strategies and an even larger range of available implementations.\nWhile it is unreasonable to expect a full review of the available methods, the inclusion of even a single ‘large-scale’ dataset in the test set or use cases would go a long way towards demonstrating the scalability of sourmash. Selecting a biologically relevant subset from a public genomic repository such as the NIH SRA, TCGA, or GTEx (to name just a few) would alleviate the need to host such a dataset while allowing large-scale reproducibility and benchmarking.\n\nMinor Comments:\nThe ‘Salmon & edgeR’ MDS plot in Figure 1 does not have points associated with the text labels. As there is no consistency in the placement of labels versus nodes in the first plot (Sourmash Compare MDS), even approximate values are difficult to determine.\n\nThe commands listed in the manuscript are using the wrong character (‘–’ vs ‘-’). None of them run properly without manual adjustments or retyping. I imagine this is a formatting issue more than a coding one but it would make reproducing the results a lot simpler if it was resolved.\nOther:\n\nThe inclusion of limited abundance information is a particularly interesting improvement over standard MinHash sketches. The manuscript suggests that the abundance tracking can play a significant role when ‘comparing many signatures’ but there is no concrete claim to assess. While outside the scope or focus of this work, a follow-up piece which explores the theoretical or practical impact of systematically sub-sampled counting information would be potentially high impact.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "52218", "date": "02 Sep 2019", "name": "Rayan Chikhi", "expertise": [ "Reviewer Expertise Algorithms and data structures for sequence bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present sourmash 2, a tool that implements a novel combination of SBTs and MinHashes, which are both fascinating computational concepts; thus, their mix is quite an interesting one. Sourmash 2 enables to perform large-scale sequences-vs-database similarity searches. The article offers a comprehensive guide for many of the software features, with biologically relevant scenarios. This is a useful contribution that is highly relevant to current needs in biology. There are a few technical issues with the current manuscript version that I list below. But otherwise, most of my remarks are for adding some extra perspective. I believe the manuscript can be approved after the technical fixes.\nMajor remarks:\nA quick recap of the state of the art in containment search would be helpful. Here you claim to use ‘a modulo approach’. Mash screen and containment minhash use different approaches (see e.g. the blog post of ‘Mash screen’). It would be nice if, in this paper, the usage of the modulo approach was put into perspective compared to those two aforementioned methods.\n\nIn fact, in the blog post cited as reference 8, Ondov writes that “the modulo approach is problematic for metagenomic applications (e.g. finding a virus in a metagenome).” The problem is indirectly mentioned in the manuscript (“can sacrifice some of the memory and storage benefits of standard MinHash techniques, as the signature size scales with the number of unique k-mers”). It would be neat to get the authors’ comparative perspective here as to why using modulo is the better approach.\n\nMy main comment would perhaps be the lack of comparison with other software. I do not know if this is a requirement for F1000Research in the “Software Tool Article” category. I suppose that sourmash is the only tool that implements SBT-Minhashes, so of course here there is no competitor in that category. It would however be nice to have some indication on whether sourmash is best-in-class in each of the proposed features (the uses cases), or whether other tools already exist and somehow do a similar job. And, the other way around, which areas where sourmash is really the only tool capable of doing X in reasonable time. I do not expect a comprehensive benchmark, but some informal indication would already be appreciated.\n\nWhat are roughly the limits of similarity queries? E.g. sequences shorter than X or having identity below Y% have no chances to be reported.\n\nA summary of all the features demonstrated in the main text could be helpful. For instance, reading only the introduction, it is not explicit that a natural application of sourmash is outlier/contaminant detection.\n\nMinor remarks:\n“Sequence Read Archive now contains over 20 Petabases of data1”: seems to be over 30 PB in 2019 according to the plot in reference 1.\n\nIt is not clear what the ‘LCA’ term stands for in the context of the database format introduced here. Is it the lowest common ancestor?\n\nThe description of LCA (in section “LCA database”) is imprecise. What does a “named list” mean in this context? A Figure would be helpful to see a small example.\n\nA sentence in the manuscript mentions ‘a second database format’. The ‘first format’ is supposedly the SBTMH but it is only implicit that SBTMH is a ‘format’.\n\nThe introduction mentions a bunch of features implemented in other tools (“Jaccard similarity comparisons, .., k-mer abundance comparisons, decrease runtime and memory requirements, and work on streaming input data.”) Are all of these implemented in sourmash2, or only a subset of them? (It seems to me that most are implemented.)\n\nThe description of the modulo approach used is imprecise. How is the hash space divided into s equal ‘bands’ (undefined term), precisely? Also, I suppose this somewhat different from the modulo approach proposed by Broder, and clarified in Mash screen’s blog post, but how so?\n\nThe concept of ‘hash subset retention’ is not well defined. I suppose it is the set of hashes that result from a MinHash computation.\n\nAbundance filtering (as in Finch) is not performed in sourmash2, right?\n\nSome of the ‘signature utilities’ are self-explanatory. However, what is the difference between ‘intersect’ and ‘overlap’? What is ‘flatten’?\n\nRegarding the sentence: “although we recommend using more accurate approaches for detailed comparisons.” To make the paper self-contained, a short explanation would be needed to delineate what sort of concrete use-case(s) is/are meant behind the term ‘detailed comparisons’.\n\nThe “similarity queries” and “containment queries” sections could benefit from at least one use-case example per query. This is to illustrate the two sections, which are a bit obscure without examples. (I realize that use cases are given later in the codes examples, so perhaps a forward-reference could work, albeit less elegant.) A proposal for similarity queries: ‘find all genomes in the SBTMH (leaves) that are similar to a query genome’.\n\nAwkward formulation of that part of the sentence: [..] ‘using a k-mer size of 4, use all 4-mers, and track abundance.’ (although I understood that the k-mer size was 4 and the ‘use all k-mers’ refers to a scaling factor of 1)\n\nThe “Tetranucleotide Frequency Clustering“ section is quite nice. It should be emphasized however this isn’t really a minhash sketch: all 4-mers are considered.\n\nRegarding the sentence “We see that the minimum similarity in the matrix is 0%”, how is that seen? visual inspection of ecoli.comp.matrix.png?\n\nRegarding the sentence “This process is repeated until the threshold is reached.”: I forgot.. which threshold?\n\nRegarding the sentence “We see that 20.1% of k-mers match 82 genomes in GenBank.”: how is this seen? Also “we see two matches between P. ruminis strains among all matches.” In the output above that text, I see only one match. (I could not test that section due to the missing download URL.)\n\nRegarding the software commands:\nAs an important note, one cannot easily copy-paste the command lines as short dashes (-) are converted to long dashes (–). Nevertheless, I still automatically replaced all the dashes and tested all command lines. I’ll report any problem below.\n\nExtra ‘\\’ at the end of the command: “curl –L –o ecoli–reads.khmer.fq.gz\n\nhttps://osf.io/26xm9/download \\”.\n\nMissing url in command (and also extra ‘\\’ at the end): “[..] unmapped–qc–to–ref.fq.sig https://osf.io//download \\”\n\nThus I could not test this part.\n\nThe streaming operation at the beginning of the MDS section overwrites the file ‘ERR458584.khmer.sig‘ produced before, perhaps make a note of that.\n\nThe url “curl –L –o genbank–d2–k31.tar.gz \\\n\nhttps:// s3–us–west–2.amazonaws.com/\n\nsourmash–databases/2018–03–29/\n\ngenbank–d2–k31.tar.gz“ has extra new lines\n\nIn the command “sourmash index –k 31 ecolidb \\\n\nescherichia–sigs /*.sig”, a space is wrongly inserted after “escherichia-sigs”\n\nAnd also, in the command that follows, one of the ‘\\’ is extra and another ‘\\’ is missing.\n\nThe SBT path inside the file ‘fungi-k31.sbt.json’ is wrongly hardcoded. Also, when fixing it, I get “WARNING: this is an old index version, please run `sourmash migrate` to update it.” Although it did end up working.\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": [] } ]	1	https://f1000research.com/articles/8-1006
https://f1000research.com/articles/8-420/v1	09 Apr 19	{ "type": "Study Protocol", "title": "The effects of auditory spatial training on informational masking release in elderly listeners: a study protocol for a randomized clinical trial", "authors": [ "Farnoush Jarollahi", "Marzieh Amiri", "Shohreh Jalaie", "Seyyed Jalal Sameni", "Farnoush Jarollahi", "Shohreh Jalaie", "Seyyed Jalal Sameni" ], "abstract": "Background: Regarding the strong auditory spatial plasticity capability of the central auditory system and the effect of short-term and long-term rehabilitation programs in elderly people, it seems that an auditory spatial training can help this population in informational masking release and better track speech in noisy environments. The main purposes of this study are developing an informational masking measurement test and an auditory spatial training program. Protocol: This study will be conducted in two parts. Part 1: develop and determine the validity of an informational masking measurement test by recruiting two groups of young (n=50) and old (n=50) participants with normal hearing who have no difficulty in understanding speech in noisy environments. Part 2 (clinical trial): two groups of 60-75-year-olds with normal hearing, who complain about difficulty in speech perception in noisy environments, will participate as control and intervention groups to examine the effect of auditory spatial training. Intervention: 8 sessions of auditory spatial training. The informational masking measurement test and Speech, Spatial and Qualities of Hearing Scale will be compared before intervention, immediately after intervention, and one month after intervention between the two groups. Discussion: Since auditory training programs do not deal with informational masking release, an auditory spatial training will be designed, aiming to improve hearing in noisy environments for elderly populations. Trial registration: Iranian Registry of Clinical Trials (IRCT20190118042404N1) on 25th February 2019.", "keywords": [ "Informational masking", "Energetic masking", "Elderly", "Train", "Speech perception in noise" ], "content": "Introduction\n\nUnderstanding speech in noisy environments is a major challenge of the auditory system, which occurs mostly due to aging. It is clear that poorer speech recognition in the elderly population can occur due to various factors, including peripheral hearing impairment or decline in cognitive capabilities and processing defects at supra threshold levels. It is difficult to determine the exact role of each of these factors in developing speech problems in the elderly1. Most elderly individuals complain about the difficulty in understanding speech in noisy environments, despite having normal hearing thresholds2. In addition, the type of background noise heavily influences the extent of the damage imposed to speech intelligibility1. Generally, hearing problems worsen in noisy environments when the target speech is covered by a competing signal. In this situation, in addition to energetic masking, there is another type of masking known as informational masking3.\n\nThe spectro-temporal overlap between target and competing speech, which leads to poor target identification, is called energetic masking4. Energetic masking is caused by physical interactions between target and competing speech5,6 at the low level of the peripheral auditory system7. Recent research has suggested that when competing signals occur randomly or when there is a high similarity between target and competing signals (for example, when both signals are speech), another type of masking occurs both. This type of masking, which occurs in response to uncertainty of the competing signal or similarity between target and competing signals, is called non-energetic or informational masking8,9. It leads to failure in selecting auditory objects and therefore impairs auditory scene analysis. Generally, in contrast with the energetic masking, which occurs due to the limitations caused by frequency selectivity at peripheral levels, informational masking reflects the processing capacity limitations at central auditory levels10.\n\nVarious studies have indicated that with an increase in age, side-effects of competing noises will increase1,3,11,12. Different studies have revealed that elderly populations, who do not have peripheral auditory impairment, suffer from diminished ability of using acoustic and phonetic signs to separate speech from background noise, compared to young people; therefore, more informational masking occurs in this population3,13. Since problems with understanding speech will reduce social interactions of the elderly population, it is very important to develop effective auditory rehabilitation programs to prevent their isolation and to improve their quality of life1.\n\nAuditory spatial processing plays an important role in speech recognition in complex noisy environments14, since it enables the listener to differentiate the target signal from competing signals via auditory scene analysis and forming auditory streams15. Based on the results of different studies, it has become clear that the most important sign of informational masking release is spatial separation of target and competing signals16. In addition, it has been shown that auditory spatial processing ability is lower in the elderly with normal hearing than in young people. The reduction of localization accuracy and taking advantage of auditory spatial processing, consequently decreasing binaural processing, are not totally related to impaired hearing thresholds17,18. Hence, it leads to poorer speech recognition in elderly people with normal hearing in noisy environments14. On the other hand, it has been shown that the elderly population need a higher signal to noise ratio for speech recognition in the presence of noise, compared to young people. These changes are possibly due to the reduction in the ability of using acoustic and phonetic signs to separate target signals from background noise11. Therefore, in elderly people, without considering the hearing impairment, the ability to use spatial and non-spatial signs for informational masking release diminishes due to the reduction of cognitive processing abilities11,12, temporal processing defects3, defects in the connection between hemispheres, and diminished ability to separate simultaneous sounds11.\n\nCurrent neuroscientific studies have suggested that the central auditory system has a strong neuroplasticity capability for auditory spatial processing19,20 and since the effect of short-term and long-term auditory rehabilitation programs has been demonstrated in elderly people2, it seems that by providing auditory spatial training, we can aid the elderly population to perform informational masking release, preventing them from missing conversations in noisy environments.\n\nThe present study had two parts. The first part of this research was developing a test for measuring and evaluating the informational masking. The second part of this research was a clinical trial of an auditory spatial training program in elderly people with normal hearing, which could diminish informational masking. The main hypothesis for the second part of the study is that presenting an auditory spatial training for elderly people would be effective in the improvement of speech recognition in noisy environments by stimulating the centers related to binaural processing.\n\n\nProtocol\n\nThis is version 1 of this protocol.\n\nThis research will be conducted in the Audiology Clinic of Rehabilitation Faculty of Iran University of Medical Sciences.\n\nThis study consists of two main parts. Part 1: develop and determine an informational masking measurement test and explain its validity characteristics in a test development study, conducted cross-sectionally. The study population will be a group of elderly (60 to 75 years old) and a group of young (20 to 40 years old) people. The young people will be recruited from rehabilitation students of Iran University of Medical Sciences, while elderly people are those referred to the audiology clinics of Iran University of Medical Sciences.\n\nPart 2 (simple randomized clinical trial): the effect of training on informational masking release. This part of study is a simple randomized clinical trial design and patients will be randomly assigned into two groups of control (not receiving auditory spatial training) and intervention (receiving auditory spatial training). The random allocation will be performed based on balanced randomization [1:1] where the allocation will be applied by random number table (those assigned an odd number, control group; those assigned an even number, intervention group). This allocation sequence will be generated by one of the audiology clinic staff of the IUMS who will not have any role in the study. An elderly population, 60–75-years-old, who are referred to the audiology clinics of Iran University of Medical Sciences will be selected. The two groups will be matched for age and gender. Those in the control group will not receive any rehabilitation programs during the study.\n\nInclusion criteria (for all participants in the study): auditory thresholds ≤25dB within the 250–2000Hz frequency range and <40dB at 4000Hz frequency, ensuring lack of salient cognitive problems using Mini Mental State Examination (MMSE)21; having diploma or higher degree; right-handedness (using Edinburgh handedness inventory); speaking Farsi and being monolingual; complaint about speech in noise perception difficulties (just for those in part 2 of the study); and normal condition of middle ear function.\n\nExclusion criteria (for all participants in the study): unwillingness for participation in each step of research and not meeting inclusion criteria.\n\nPart 1: Developing an informational masking measurement test and determining its validity. When studying the informational masking, use of the coordinate response measure (CRM) has been frequently been introduced as one of the most popular speech materials for evaluating informational masking22. In these sentences, the same rigid structure with \"Ready [call sign] go to [color] [number] now\" format is used. In these sentences, eight call signs, four colors, and eight numbers from 1 to 9 can be used. These sentences will be expressed by speakers of different genders22–24. In the present study, 256 sentences will be created for each speaker (848). Sentences will be expressed by four speakers (two women and 2 men), providing a total of 1024 sentences. Although CRM stimuli have been initially designed to measure the speech perception in the presence of competing signals, these speech materials provide no contextual information; i.e. predicting the given color or number in the phrases is not possible. This is an important factor in measuring informational masking23.\n\nSince there is no Persian version of these sentences, this research will prepare the sentences and determine their content and face validity and reliability. Then, after selecting the nouns, colors, and numbers used in the sentence, conforming to the main English version, the prepared sentences will be given to experts in this field (audiologists, speech therapists, and linguistics) to determine the content validity. These experts are the academic members of rehabilitation faculties of IUMS, Tehran University of Medical Sciences (TUMS) and Shahid Beheshti University of Medical Sciences (SBUMS). These experts will be emailed a questionnaire to score the validity items (see Table S1, Extended data).\n\nAfter selecting the best pattern matching Persian language, based on the model presented for recording the sentences, all sentences will be recorded in a studio with the four speakers. In order to record the sentences, all criteria of the English version including the sampling rate of 44.1 kHz and giving 3s to speakers to produce each sentence will be followed. Then all the sentences will be scaled and all the words in CRM will be set such that they occur simultaneously, called coordinate sentences23 where each of the sentences will be filtered using a band pass filter of 80 to 8000 Hz filter. Again, in order to determine the face validity, the recorded sentences will be given to the experts mentioned above to determine their suitability. They must fill the questionnaire, which will be emailed to them.\n\nTo determine the reliability of CRM speech materials, the mean scores of CRM recognition in the silent will be evaluated in a group of young and old people with normal hearing who do not have speech perception difficulties in noisy environments. There will be one preliminary test and then a re-test. This evaluation will be implemented at the comfort hearing level of the participant. The score will be calculated based on the correct recognition percentage of the sentences. A sentence will receive a correct score when the color-number combination recognition is recognized correctly23,24. In this study, the mean correct score for color, number, and noun will be studied separately in order to calculate the error percentage for each of them22. By preparing these sentences, they can be used in the part 2 of the study.\n\nThe best way to measure informational masking value is determining the score for speech recognition in the presence of meaningful and meaningless competing noise. To this end, the recognition score for Persian version of CRM speech corpus will be measured under two conditions:\n\nA. In the presence of meaningful competing noise: The competing signal is selected from the Persian version of CRM corpus, where the call sign, color and number used in the competing sentence is different from those of the target sentence and it will be expressed by a different speaker. The individual will be trained to pay attention to certain target call signs and ignore other signals22–24. As one of the important effective factors for informational masking is the great similarity between target and competing signals (like when both of them are speech)8,9, using CRM sentences as both the target signal and competing signal, the high semantic and syntactic similarity would develop between target and competing signals22,23.\n\nB. In the presence of meaningless competing signal: the previous signal is manipulated such that its spectrum content remains fixed but meaningless - indeed, energetic masking remains but informational masking is reduced. \"Time-reversed speech\" will be used for this purpose. This is one of the most effective methods in behavioral and neurophysiological research performed for the effects of speech signals on each other. In this method, by fixing the long-term acoustic spectra of two signals and manipulating one of them such that it divides into non-overlapping time segments, and with reverse time presentation of each segment connecting them to each other, we will have a signal which is equal with the first signal in terms of the spectrum but is not understandable25. In the case of using 20–40 millisecond time windows, this method does not have significant effectiveness in non-understanding the speech signal; therefore, longer time windows should be used25. MATLAB R2018 software will be used in constructing this signal.\n\nThe signal to noise ratio in sentence recognition test in steps A and B was ±10, ±5, and 0. The target signal is always presented from a loudspeaker in the 0-azimuth degree and two competing signals from the loudspeakers, which are at ±90 degree and 0 azimuth degree (once with spatial separation and once in the direction of target signal), where once the competing signal has the same gender as the target signal and another time has a different gender. As a result, 20 conditions will be evaluated at each step (5 signal to noise ratios and two spatial angles with two different genders).\n\nFinally, the informational masking score in all 20 conditions will be calculated as follows:\n\nSpeech recognition score in the meaningful competing noise condition-speech recognition score at non-understandable noise condition=informational masking score (percentage).\n\nIn this step of the research, construct validity will be used to determine the validity of the test. For this purpose, Speech, Spatial, and Qualities of Hearing Scale (SSQ) questionnaire score of each individual will be compared against the informational masking score26. Figure S1 (Extended data) represents the participant’s timeline of the first part of the study.\n\nPart 2: the effect of auditory spatial training on the informational masking release. This part of research will be conducted in three steps: before auditory spatial training, during training, and after training.\n\n1. Assessments prior to auditory spatial training (preliminary interview)\n\n- Obtain patient history to confirm the inclusion and exclusion criteria of the participants\n\n- Initial clinical examination, including otoscopy and tympanometry\n\n- Perform pure tone audiometry test\n\n- Perform MMSE questionnaire to ensure lack of salient cognition problem in the participants21\n\n- Determine speech perception difficulties in the presence of noise: this was evaluated with a question: Do you have difficulty in understanding speech in noisy situations? There were three response options: yes, no or sometimes. Those who responded yes were entered into the study.\n\n- Measure informational masking score using the test constructed in Part 1 (primary outcome)\n\n- Determine the SSQ score26. The SSQ self-assessment questionnaire will be filled out by the researcher during the preliminary interview. As improving informational masking can improve the quality of life of people, this questionnaire will be used to measure the quality of life of the participants quantitatively (secondary outcome).\n\n2. Providing auditory spatial training (intervention group only)\n\nAuditory spatial training is designed based on five signs that are important in informational masking release: angular differentiation between target and competing signals16,27–29; signal to noise ratio27; similarity and difference between the target and competing signals12; similar or different gender for target and competing signals12,30; and meaningfulness of the competing signal12. Training sessions will be divided into three general steps by considering the competing signals. In the first step, meaningless competing signals will be used like white noise. In the second step, in order to make the training process somewhat difficult, meaning-carrying signals like speech babble consisting of four speakers will be used. Finally, sentence materials with male and female genders will be used. The reason for using the gender factor is that consideration of gender similarity or difference between target and competing signals is one of the signs that adults use for informational masking release12,30.\n\nIn all steps, the target signal will be presented from the loudspeaker at 0-azimuth degree and competing signals from different azimuth angles such as ±90 and 031,32. Therefore, the difficulty of training will grow in each step by reducing the azimuth angle of the competing signal33.\n\nSentence signals are selected from Persian version of QuickSIN34. Every step of training is implemented as follows:\n\nThe intensity of the competing signal is fixed at 60 dBSPL, and at the beginning the intensity of target signals is 70 dBSPL. Three first sentences are used for familiarization. If an individual needs more practice, more familiarization sentences are provided.\n\nAn individual is requested to identify the keywords heard in the target sentences. In the case of true and false identification, required feedback is provided. If the individual identifies more than 50% of the keywords, the sentence is considered true. In this signal to noise ratio, 5 sentences are provided where if the individual identifies more than 50% of the presented sentences, the signal to noise ratio decreases in 5dB steps, after which 5 sentences will be provided again for the individual. If the individual does not have the capability to correctly identify more than 50% of the presented sentences in each signal to noise ratio, the training begins where this signal to noise ratio will be considered as the initial level. The training will continue for 20 minutes and the intensity will change in an adaptive procedure, such that in the case of correct identification of a sentence, the intensity will be decreased by 1.5dB while it will be increased by 2.5 dB if the individual scored less than 50% of words correctly. At each intensity where an individual can correctly identify the sentences, the next sentence will be presented and the above process will repeat.\n\nThe optimal condition for perceptual auditory learning includes active listening to high repetition of signals during the consecutive educational sessions, which is conducted within a short time interval. Since long-term training is not a very suitable option in the clinic2, trainings are repeated twice a week over 8 sessions35.\n\n3. Assessments after auditory spatial training (interview immediately after and one month after training)\n\nThe informational masking test (as per Part 1) will be done immediately after training and one month after using the Persian list of the coordinate response measure (CRM) corpus, which will be compared with the pre-training results (preliminary interview). This score will be the primary outcome. The reason for repeating experiments one month after the intervention is determining the reliability of the results obtained by intervention for informational masking release.\n\nThe informational masking release value will be calculated based on the difference between sentence recognition score (in all 20 conditions of signal to noise, different spatial angles, and two genders) in both noise situations (meaningful and non-understandable). The changes in informational masking in the assessments will be calculated before and after the intervention across all 20 conditions (see Table S2, Extended data).\n\nAs the ultimate purpose of this research is improving the quality of life of elderly people, the score of SSQ immediately and one month after intervention will be obtained and the results of both intervention and control groups will be compared separately. This score will be the secondary outcome of the intervention. Figure S2 (Extended data) represents participants timeline of the second part of the study.\n\nPart 1. The study of Terwee et al. was considered as the basic study to determine the sample size of the first part of our study. They suggested that at least 50 patients in each group must be included to evaluate the construct validity36. In total, 50 young people aged between 20 and 40 years and 50 elderly people aged between 60 and 75 years, with normal hearing who do not suffer from speech understanding in noisy environments, will be recruited.\n\nPart 2. The following formula is used to determine the sample size:\n\nS1: standard deviation of the studied variable in the first group (case, exposed, or intervened)\n\nS2: standard deviation of the studied variable in the second group (control, unexposed, or compared)\n\nµ1: mean of the studied variable in the first group\n\nµ2: mean of the studied variable in the second group\n\nα=0.05\n\nβ=80%\n\nIn this formula, the studied variable is the extent of informational masking changes before and after the intervention. There is no previous study, which was used the same training as this study proposes; therefore, we considered the study of Delphi et al. which was on a group of elderly individuals37. The sample size of this study was 16 patients in each group. We will use the same sample size.\n\nCentral tendency and dispersion indices (mean and standard deviation) will be used in descriptive analysis of data. In data analysis and for determining the reliability of the Persian version of the coordinate response measure (CRM) corpus, paired t-test and Pearson correlation will be used in the case of normality of data; otherwise, Spearman test will be employed, and one-way ANOVA will be utilized for determining intra-class correlation coefficient.\n\nIn part 2 of the research, repeated measures ANOVA values will be used for inter-group comparisons and two-way ANOVA will be employed for comparison among groups. SPSS software (V20.0, IBM Corporation, New York, USA) will be used for statistical data analysis and the significance level for all tests will be 0.05.\n\nThe Medical Ethics Committee of Iran University of medical sciences approved the study protocol (IR.IUMS.REC.1397.303) and the ethical principles of the ethics committee will be observed in this research. Researchers will send any amendments to the protocol in the future to the ethics committee.\n\nOne of the researchers of this study will obtain written informed consent from patients willing to participate in the trial (see Extended data). The purpose of the research and its steps will be explained for all participants before the study start. Confidentiality of data and results of tests will be ensured to participants. Participants will be made aware that they can refrain from cooperation in the study when they want. Conducting tests has no side-effects for the studied individuals and all tests and training sessions are without cost to the participants.\n\nTo promote participant retention and complete follow-up, in every training session the examiner will provide feedback to all participants and will inform them about the training progress. The researcher will ask them about the impact of training on the participant’s daily communication conversations.\n\nAll data will be entered into forms which are prepared for data collection (see Table S2; Extended data) and the participant files will be stored at study site and will be maintained in a secure place and manner. Participant files will be maintained in storage for a period of 2 years after completion of the study. Only Principal Investigators will be given access to the study data.\n\nThe study outcomes will be published through peer-reviewed journals. The data resulting from this study will be released to the audiologists and participants and the general medical community. The results of this trial will be communicated to the external funding body through a formal report. There is no limit in the publication of the trial results.\n\nThe study started in December 2018 and will continue until December 2019. To date, the enrolment of the patients has been performed and the allocation will be performed in the near future.\n\n\nDiscussion\n\nSince there is a progressive increase in elderly populations around the world, the independence of this age group has gained much attention, and Iran is no exception38. One of the most important points in independent life during aging is the capability for effective verbal communication. Unfortunately, this capability declines in elderly people, especially tracking speech in environments where several speakers talk with each other. Most elderly individuals complain that despite good hearing, they cannot understand speech in noisy environments1. Indeed, elderly people cannot use auditory spatial signs for informational masking release due to the reduction of their auditory processing and cognitive abilities11,12. Since informational masking has an important role in competing signal environments and rehabilitation programs have not considered this an important aspect of masking, designing training that can help elderly people in releasing this masking is novel. Therefore, if the main research hypothesis, i.e. auditory spatial training can improve informational masking release in the elderly people, is confirmed, by providing the therapeutic solution in this age group, a series of auditory spatial trainings based on informational masking release will be provided for audiologists. In addition, the Persian version of the coordinate response measure (CRM) corpus and its reliability and validity will be calculated to be used in research on speech recognition in noisy environments.\n\n\nData availability\n\nNo data is associated with this article.\n\nOpen Science Framework: The effects of auditory spatial training on informational masking release in elderly listeners: a study protocol for a randomized clinical trial. https://doi.org/10.17605/OSF.IO/SDEJP39.\n\nThis project contains the following extended data:\n\nTable S1: The questionnaire of content and face validity of Persian version of coordinate response measure (CRM) corpus\n\nTable S2: The data collection sheet for the second part of the study\n\nFigure S1: Participant timeline for part 1 of the study (Developing an informational masking measurement test and determining its validity)\n\nFigure S2: Participant timeline for part 2 of the study (the effect of auditory spatial training on the informational masking release)\n\nInformed consent form for the participants of the first part of the study\n\nInformed consent form for the participants of the second part of the study\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nOpen Science Framework: SPIRIT checklist for ‘The effects of auditory spatial training on informational masking release in elderly listeners: a study protocol for a randomized clinical trial’: https://doi.org/10.17605/OSF.IO/SDEJP38.", "appendix": "Grant information\n\nThis study was part of a Ph.D. Dissertation approved by Iran University of Medical Sciences (IUMS), Tehran, Iran and is financially supported by IUMS (Contract No: 97-4-6-13657).\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\nThe authors acknowledge Dr Mahnaz Ahmadi for her good guidance.\n\n\nReferences\n\nGoossens T, Vercammen C, Wouters J, et al.: Masked speech perception across the adult lifespan: Impact of age and hearing impairment. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYost WA: Spatial release from masking based on binaural processing for up to six maskers. J Acoust Soc Am. 2017; 141(3): 2093–2106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDubno JR, Ahlstrom JB, Horwitz AR: Binaural advantage for younger and older adults with normal hearing. J Speech Lang Hear Res. 2008; 51(2): 539–56. PubMed Abstract \| Publisher Full Text\n\nGlyde H, Buchholz JM, Dillon H, et al.: The importance of interaural time differences and level differences in spatial release from masking. J Acoust Soc Am. 2013; 134(2): El147–52. PubMed Abstract \| Publisher Full Text\n\nShinn-Cunningham B: Models of plasticity in spatial auditory processing. Audiol Neurootol. 2001; 6(4): 187–191. PubMed Abstract \| Publisher Full Text\n\nSweetow RW, Sabes JH: The need for and development of an adaptive Listening and Communication Enhancement (LACE) Program. J Am Acad Audiol. 2006; 17(8): 538–58. PubMed Abstract \| Publisher Full Text\n\nAnsari NN, Naghdi S, Hasson S, et al.: Validation of a Mini-Mental State Examination (MMSE) for the Persian population: a pilot study. Appl Neuropsychol. 2010; 17(3): 190–5. PubMed Abstract \| Publisher Full Text\n\nBrungart DS: Informational and energetic masking effects in the perception of two simultaneous talkers. J Acoust Soc Am. 2001; 109(3): 1101–9. PubMed Abstract \| Publisher Full Text\n\nBrungart DS, Simpson BD, Ericson MA, et al.: Informational and energetic masking effects in the perception of multiple simultaneous talkers. J Acoust Soc Am. 2001; 110(5 Pt 1): 2527–38. PubMed Abstract \| Publisher Full Text\n\nBolia RS, Nelson WT, Ericson MA, et al.: A speech corpus for multitalker communications research. J Acoust Soc Am. 2000; 107(2): 1065–6. PubMed Abstract \| Publisher Full Text\n\nUeda K, Nakajima Y, Ellermeier W, et al.: Intelligibility of locally time-reversed speech: A multilingual comparison. Sci Rep. 2017; 7(1): 1782. Publisher Full Text\n\nGatehouse S, Noble W: The Speech, Spatial and Qualities of Hearing Scale (SSQ). Int J Audiol. 2004; 43(2): 85–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlyde H, Buchholz J, Dillon H, et al.: The effect of better-ear glimpsing on spatial release from masking. J Acoust Soc Am. 2013; 134(4): 2937–45. PubMed Abstract \| Publisher Full Text\n\nLin G, Carlile S: Costs of switching auditory spatial attention in following conversational turn-taking. Front Neurosci. 2015; 9: 124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreyman RL, Balakrishnan U, Helfer KS: Effect of number of masking talkers and auditory priming on informational masking in speech recognition. J Acoust Soc Am. 2004; 115(5 Pt 1): 2246–56. PubMed Abstract \| Publisher Full Text\n\nBrungart DS, Simpson BD: The effects of spatial separation in distance on the informational and energetic masking of a nearby speech signal. J Acoust Soc Am. 2002; 112(2): 664–76. PubMed Abstract \| Publisher Full Text\n\nCameron S, Glyde H, Dillon H: Efficacy of the LiSN & Learn auditory training software: randomized blinded controlled study. Audiol Res. 2012; 2(1): e15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlyde H, Cameron S, Dillon H, et al.: Remediation of spatial processing deficits in hearing-impaired children and adults. J Am Acad Audiol. 2014; 25(6): 549–61. PubMed Abstract \| Publisher Full Text\n\nTyler RS, Witt SA, Dunn CC, et al.: Initial development of a spatially separated speech-in-noise and localization training program. J Am Acad Audiol. 2010; 21(6): 390–403. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShayanmehr S, Tahaei AA, Fatahi J, et al.: Development, validity and reliability of Persian quick speech in noise test with steady noise. Aud Vest Res. 2015; 24(4): 234–244. Reference Source\n\nHumes LE, Kinney DL, Brown SE, et al.: The effects of dosage and duration of auditory training for older adults with hearing impairment. J Acoust Soc Am. 2014; 136(3): EL224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerwee CB, Bot SD, de Boer MR, et al.: Quality criteria were proposed for measurement properties of health status questionnaires. J Clin Epidemiol. 2007; 60(1): 34–42. PubMed Abstract \| Publisher Full Text\n\nDelphi M, Lotfi Y, Moossavi A, et al.: Envelope-based inter-aural time difference localization training to improve speech-in-noise perception in the elderly. Med J Islam Repub Iran. 2017; 31: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadoughi F, Shahi M, Ahmadi M, et al.: The Comparison of the Minimum Data Set for Elderly Health in Selected Countries. Acta Inform Med. 2015; 23(6): 393–397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmiri M: The Effects of Auditory Spatial Training on Informational Masking Release in Elderly Listeners: A Study Protocol for a Randomized Clinical Trial. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/SDEJP" }	[ { "id": "47087", "date": "23 Apr 2019", "name": "Frederick J. Gallun", "expertise": [ "Reviewer Expertise Auditory processing", "informational masking", "perceptual learning and training" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, we think it is an interesting study and the goal of creating a Persian version of the CRM and spatial release tests is a good idea. However, the methods for developing the new test create opportunities for confounds and difficulties interpreting the data. Specific comments are listed below. In addition, the training arm is not sufficiently different from the new informational masking for it to be a clinically reliable outcome measure. It would be appropriate to have another task (such as dichotic sentence identification (or equivalent test that has already been validated in the Persian language)) that wasn't trained as an outcome. Furthermore, the lack of an active control condition raises the possibility that any activity involving remembering and responding to stimuli (or even just coming into the testing environment) would change performance. For detailed discussion of the training issues, the reader is encouraged to consult Green et al. (2019)1. Additional fundamental methodological concerns involve the failure to consider the working memory and attention influences that are known to be important for speech in noise for older listeners (Fullgrabe et al., 2015)2 and for all listeners for informational masking tasks, especially the CRM tasks.\n\nWith the regard to the new CRM test, it is excellent to create a Persian version, but there are several differences in these methods that could result in substantial differences in the outcomes.\n\nThe step size of 5 dB is too large, given the differences in performance that are usually observed. It might be acceptable if a psychometric function were being fit to the data, but the statistical analysis proposed is unlikely to be sufficiently sensitive to the small changes that this method is able to detect, using the standard methods. A more appropriate measure is the target-to-masker ratio at which a fixed level of performance is obtained. For examples of the differences in target-to-masker ratio commonly observed in older and younger listeners with the English version for same and different-gender targets and maskers, the reader is directed to Marrone et al. (2008b)3 and Gallun et al. (2013)4.\n\nThe use of two male and two female speakers is not sufficient to ensure that the specific speaking styles of the talkers are not influencing the results. English CRM uses four of each gender, and the studies from our lab exclude one of the males due to differences in rate of speaking. Time alignment of the keywords is an essential aspect of creating an informational masking situation where spatial cues can provide large release from masking. Temporal overlap should be carefully examined, and preliminary testing should establish that none of the talkers is more intelligible than the others in the conditions to be tested.\n\nThe use of 90 degrees of spatial separation is large enough that it is possible that changes in spatial ability will not be detected. Multiple studies have established that the difference between 30 and 45 degrees of separation is not very large (Marrone et al., (2008a)5; Jakien et al., (2017)6) and that for people with normal hearing, the effects of age are difficult to detect with separations greater than 15 degrees (Srinivasan et al., (2016)7). For this study, this is very important, because if the effects of the training are to improve the ability to use (or even perceive) spatial differences, it is unlikely that this will be a very large change, and so if only very large separations are used, there may be no way to observe the improvements in performance. Jakien and Gallun (2018)8 provided mathematical equations by which the effects of age can be predicted for 45 degrees of separation. It would be useful to develop similar equations in Aim 1, compare them with the published equations, and use these normative functions to assess improvements in performance after training.\nThis report was written by Frederick Gallun with advice from Aaron Seitz.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4729", "date": "08 Jul 2019", "name": "Marzieh Amiri", "role": "Author Response", "response": "Reviewer 1Dear Dr, Gallun,We appreciate you for your complimentary comments and suggestions. The followings are our point-by-point responses:1 Your concerns about CRM were as bellow: 1) The step size and use a target-to-masker ratio.Response: We have revised this part according to Marrone et al.’s study. You can find it in the revised manuscript (Study procedure, Part1, the last paragraph).2) The number of speakers:Response: you are totally right and we will use eight speakers for recording the CRM phrases and we have revised that in the manuscript. 3) The use of 90 degrees of spatial separation is large enough that it is possible that changes in spatial ability will not be detected.Response: Your comment is very useful. We have added the ±45 degree to the study and we have revised the methodology according to your concerns. We also will use the mathematical equations which you mentioned to evaluate the effects of age.2 Your concerns about training were as bellow:1 The training arm is not sufficiently different from the new informational masking for it to be a clinically reliable outcome measure. It would be appropriate to have another task (such as dichotic sentence identification (or equivalent test that has already been validated in the Persian language)) that wasn't trained as an outcome.Response: We also have added the ‘Farsi language version of synthetic sentence identification test’ in pre and post- training parts of the study. We must mentioned that the ‘dichotic sentence identification test’ have not been converted to Persian. The original manuscript was revised accordingly.2 Furthermore, the lack of an active control condition raises the possibility that any activity involving remembering and responding to stimuli (or even just coming into the testing environment) would change performance.Response: This condition is the same for both groups. So we assumed that both of them may learn or remember the tests items. So the differences between them will be diminished. Also as you know the CRM phrases are contextual-free and it is a good point of them. It means that being in the test environment have not any large effect on scoring the test and anyone who participated in this study must just rely on her or his audibility to identify the correct color and number.3 Failure to consider the working memory and attention influences.Response: As you correctly concerned about the working memory effects, we have added a ‘Persian Reading Span test’ to evaluate the working memory ability of the participants. We will try to match people of both groups according to their scores on this test and the other tests which mentioned in the text in pre and post training situations. We must mentioned that we also use the MMSE questionnaire to include the persons who have no salient cognition impairment." } ] }, { "id": "46974", "date": "20 May 2019", "name": "Nehzat Koohi", "expertise": [ "Reviewer Expertise Assessment and management of auditory processing deficit in patients with neurological disorders. Diagnostic audiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 has two aims: first developing and validating a test to measure informational masking and second to conduct a randomised clinical trial to evaluate the effectiveness of an auditory training in elderly people who have speech in noise difficulty. The introduction is well-written, and the aims are clearly stated however the methodology needs to be revised. The detailed comments are as below:\nIntroduction\nFirst Paragraph\n‘Understanding speech in noisy environments is a major challenge of the auditory system, which occurs mostly due to aging.’ Please revise this sentence. There are many reasons for difficulty understanding speech in background noise, aging is one of them, it doesn’t occur mostly due to aging. Please first define/explain what energetic and informational masking are (please re-organise your paragraphs, the definition should come first)\n\nSecond paragraph\n‘…, another type of masking occurs both.’ Please revise this sentence, it doesn’t make sense.\n\nFourth paragraph\n‘Based on the results of different studies, it has become clear that the most important sign of informational masking release is spatial separation of target and competing signals’. Here you’ve mentioned several studies but only used one reference, either revise the sentence or add more references.\n\nFifth paragraph\n‘On the other hand, it has been shown that the elderly population need a higher signal to noise ratio for speech recognition in the presence of noise, compared to young people.’ Please add a reference. ‘in elderly people, without considering the hearing impairment, the ability to use spatial and non-spatial signs for informational masking release diminishes due to the reduction of cognitive processing abilities, temporal processing, defects in the connection between hemispheres, and diminished ability to separate simultaneous sounds.’ By ‘hearing impairment’ do you mean apparent hearing loss on the PTA? Hearing impairment includes temporal (and auditory) processing deficits as well as peripheral hearing impairment.\n\nLast paragraph\n‘The present study had two parts. The first part of this research was developing a test for measuring and evaluating the informational masking.’ The sentences are in the past, have you done the study already or you are going to conduct these? In the study design section, you mention ‘the study will be conducted...’ Please be consistent. Although you have clarified this in your methodology, your aim will be clearer if you specify here why you are developing and validating a test to measure informational masking.\n\nStudy design\n‘Inclusion criteria (for all participants in the study): auditory thresholds ≤25dB within the 250–2000Hz frequency range and …’ My main concern is the definition of ‘normal hearing’. Which protocol have you used to define ‘normal hearing’? if you are including patients with mild high frequency hearing loss you cannot say patient’s hearing is normal. Even if all frequencies (250-8000 Hz) are better than 20 or 25 dBHL (BSA or ASHA definition) if the OAEs are not robust or wave I of the ABR is absent you cannot assume patient’s hearing is normal. Pure tone audiogram is very limited when testing the auditory pathway. In addition, you need to differentiate between peripheral and central hearing. If you would like to use the term normal hearing, it’s better to say ‘peripheral hearing’ as you have not tested the central auditory pathway so you cannot assume their hearing is ‘normal’. The prevalence of hearing impairment (peripheral and central) in elderly people is quite high. Also it is perfectly possible that an elderly patient has a spatial processing disorder in the presence of 'normal pure tone audiogram'. SSQ is not a quality of life questionnaire, please use another term and revise this paragraph. Please justify why you chose 4 speakers.\n\nSample size\nPlease specify what Z in the sample size formula is and include means and standard deviations from the Delphi’s study. The sample size for study 2 is not adequately justified. Sixteen participants seems a very low number, particularly if we assume 20% drop out. How many participants are you going to approach?\n\nOther comments\nSufficient time must be considered for auditory training, one month seems a very short time to assume auditory training is beneficial or not (in Humes et al’s methodology the training was performed twice a day for 7.5 weeks but you are proposing 2 trainings in 4 weeks) , ideally you should repeat your outcome measures after 3 and 6 months, and even better if you could repeat 1 month after the training is ended to explore the long term potentiation. Feedback and monitoring are crucial in auditory training, you have mentioned about the feedback but please explain in detail how this will be done. There was no mention of progression in difficulty of the training (one of the main principles of auditory training), are you going to consider this in your training? Since you have not done any pilot or feasibility study, it would be useful if you could do a small qualitative study; for example, running a focus group and asking your participants what they think of the intervention and how likely they will continue performing the auditory training in the future. One of the major issues with the auditory training (or any other training) is boredom and generally keeping the patient motivated throughout. In addition, in practice, performing the training in the clinic is time consuming and costly. Would patients do these training at home? Adhering to the training while performing at home is another issue. The consent form needs to be written in lay language and please avoid jargon and acronyms.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4730", "date": "08 Jul 2019", "name": "Marzieh Amiri", "role": "Author Response", "response": "Dear Dr, KoohiWe appreciate you for your complimentary comments and suggestions. The followings are our point-by-point responses:1 Your comments about ‘Introduction’ part were:First paragraph: please revise this sentence:’ Understanding speech in noisy environments is a major challenge of the auditory system, which occurs mostly due to aging.’ And please first define/explain what energetic and informational masking are (please re-organize your paragraphs, the definition should come first)Response: As suggested by you, we have revised the first sentence to:‘Many older adults complain about speech perception in noisy situations.’ And we also re-organize the paragraph according to your comment.Second paragraph: Another type of masking occurs both.’ Please revise this sentence, it doesn’t make sense.Response: We are sorry that this part was not clear in the original manuscript. We have revised the contents of this part.Fourth paragraph: ‘Based on the results of different studies, it has become clear that the most important sign of informational masking release is spatial separation of target and competing signals’. Here you’ve mentioned several studies but only used one reference, either revise the sentence or add more references.Response: we have added more references.Fifth paragraph: ‘On the other hand, it has been shown that the elderly population need a higher signal to noise ratio for speech recognition in the presence of noise, compared to young people.’ Please add a reference.Response: we have added references.‘in elderly people, without considering the hearing impairment, the ability to use spatial and non-spatial signs for informational masking release diminishes due to the reduction of cognitive processing abilities, temporal processing, defects in the connection between hemispheres, and diminished ability to separate simultaneous sounds.’ By ‘hearing impairment’ do you mean apparent hearing loss on the PTA? Hearing impairment includes temporal (and auditory) processing deficits as well as peripheral hearing impairment.Response: Your comment is totally true. We have mentioned ‘peripheral hearing impairment’ in the sentences.Last paragraph: ‘The present study had two parts. The first part of this research was developing a test for measuring and evaluating the informational masking.’ The sentences are in the past, have you done the study already or you are going to conduct these? In the study design section, you mention ‘the study will be conducted...’ Please be consistent. Response: We have corrected the sentences.Although you have clarified this in your methodology, your aim will be clearer if you specify here why you are developing and validating a test to measure informational masking.Response: We have explained this and added more sentences to this part.2 Your comments about the ‘ study design’:‘Inclusion criteria: You need to differentiate between peripheral and central hearing. If you would like to use the term normal hearing, it’s better to say ‘peripheral hearing’ as you have not tested the central auditory pathway so you cannot assume their hearing is ‘normal’.Response: We have revised this criteria accordingly to: “Auditory thresholds ≤25dB within the 250–8000Hz frequency range for ensuring the normal pure tone audiogram or normal peripheral hearing,”SSQ is not a quality of life questionnaire, please use another term and revise this paragraph.Response: We have revised this sentence as bellow:‘As improving informational masking can improve the speech perception quality of people, this questionnaire will be used to measure the speech perception quality of the participants quantitatively (secondary outcome).’Please justify why you chose 4 speakers.Response: We have corrected this part and we will use eight speakers for recording the CRM phrases.3 Your comments about the ‘ sample size:Please specify what Z in the sample size formula is and include means and standard deviations from the Delphi’s study. The sample size for study 2 is not adequately justified. Sixteen participants seems a very low number, particularly if we assume 20% drop out. How many participants are you going to approach? Response: We have included all you mentioned in the revised version.4 Other comments you mentioned were:1) Training period which have corrected as bellow‘The optimal condition for perceptual auditory learning includes active listening to high repetition of signals during the consecutive educational sessions, which is conducted within a short time interval. Since long-term training is not a very suitable option in the, trainings are repeated three times a week completed in 5-week cycles.’2) Feedback and monitoring are crucial in auditory training, you have mentioned about the feedback but please explain in detail how this will be done.Response: We have included your comment to the text.3) There was no mention of progression in difficulty of the training (one of the main principles of auditory training), are you going to consider this in your training?Response: We have added more explanation to this part. As bellow.‘As one of the main principles of any auditory training program is progression in difficulty so the training sessions will be divided into three general steps by considering the competing signals. In the first step, meaningless competing signals will be used like white noise. In the second step, in order to make the training process somewhat difficult, meaning-carrying signals like speech babble consisting of four speakers will be used. Finally, for making more difficulty, sentence materials with male and female genders will be used. The reason for using the gender factor is that consideration of gender similarity or difference between target and competing signals is one of the signs that adults use for informational masking release.’4) Since you have not done any pilot or feasibility study, it would be useful if you could do a small qualitative study; for example, running a focus group and asking your participants what they think of the intervention and how likely they will continue performing the auditory training in the future.Response: You have mentioned a very good point and we have added your comment to the text. You can find it in the third paragraph of the ‘Ethical statement and consent to participate’ part.5) Would patients do these training at home?Response: Because we will design our study with loudspeakers it will be not done at home. But this study is kind of a pilot study and if this training will have good results maybe it would be done in the future and under headphones.6) The consent form needs to be written in lay language and please avoid jargon and acronyms. Response: We have revised it." } ] } ]	1	https://f1000research.com/articles/8-420
https://f1000research.com/articles/7-1430/v1	07 Sep 18	{ "type": "Research Article", "title": "Distal middle cerebral artery occlusion does not result in depression-like behaviours", "authors": [ "Yvonne Couch", "Bettina Hjelm Clausen", "Maria Ormhøj", "Maria Gammelstrup Andersen", "Christine Kring", "Maja Møller", "Kate Lykke Lambertsen", "Bettina Hjelm Clausen", "Maria Ormhøj", "Maria Gammelstrup Andersen", "Christine Kring", "Maja Møller", "Kate Lykke Lambertsen" ], "abstract": "Background: Stroke is a devastating neurological injury, which can result in significant cognitive and behavioural deficits. Modelling the disease processes associated with stroke in animals is key to the development of novel therapeutic approaches. However, some aspects of stroke pathophysiology, including neuropsychiatric symptoms, do not translate well from humans to animals. Here, we aimed to investigate the development of post-stroke depression in a rodent model of stroke. Methods: The distal middle cerebral artery (MCA) was permanently occluded by electrocoagulation in adult male C57/Bl6/J mice. Animals were allowed to survive for 6 hours, 24 hours, 2 days, 5 days or 7 days prior to behavioural testing. Brains were taken to confirm lesion volumes at the above times. Behavioural tests studied basic exploration and motivation (open field and marble burying) as well as depression-like behaviours (tail suspension and sucrose preference). Results: Animals developed robust and reproducible lesions in the cortex but whilst stroke reduced activity in the open field, animals showed no associated behavioural deficits in any of the tests used for depression-like behaviours. Conclusions: The distal middle cerebral artery occlusion (MCAO) model results in a small cortical lesion which produces no depression-like behaviours. These negative data are important for those wishing to investigate the more cognitive and behavioural aspects of stroke.", "keywords": [ "stroke", "MCAO", "depression", "anxiety" ], "content": "Introduction\n\nMajor depression is an important neuropsychiatric consequence of stroke, and develops in approximately one third of stroke patients, often independent of functional deficits1,2. Modelling affective deficits in rodents is key to the development of novel therapeutic strategies, but has proved challenging thus far. Rodent models of post-stroke depression often combine middle cerebral artery occlusion (MCAO) models with models of chronic stress3. Whilst this results in a semi-reproducible set of animals showing both lesions and depression-like behaviours, it is not necessarily representative of the human condition.\n\nThe majority of ischemic strokes occur in the territory of the middle cerebral artery4. As such this is most commonly modelled in rodents5. The introduction of the use of endovascular treatment for acute ischemic stroke means that the transient model of stroke has good face and construct validity6. However, model variability still blights pre-clinical stroke research and direct translation of useful therapies has been limited7,8. The permanent MCAO model results in a consistent cortical infarct which shows reproducible evolution. This model is used to mimic human stroke without reperfusion, which represents the majority of clinical strokes.\n\nThe evolution of the lesion results in both local central nervous system (CNS) inflammation and changes in systemic immune reactivity9,10. MCAO is known to induce activation of microglia outside the territory of the MCA, even into the contralateral hemisphere11, a finding which translates to human stroke where microglial activation has been found in regions distant from the core infarct12,13. Inflammation within the CNS, both from internal activation of glial cells and from infiltrating immune cells and inflammatory mediators, is a known causative factor in depressive-like behaviours, both in rodents and humans14–16. Indeed in a number of CNS diseases, including stroke, widespread inflammation and depression are now being causally linked17–19. As such, the inflammation associated with the distal MCAO model, which has also been shown to extend into the contralateral hemisphere20 could potentially result in a depression-like phenotype.\n\nStudying affect in rodents provides its own challenges, with the most common models of depression involving either transgenic animals or models of chronic stress21. Combining these approaches with models of stroke results in a rodent model which shows very little face validity, but rather a complex model of vascular disease and stress. In addition, the use of methodologies commonly used to study depression, such as the forced swim test, may be incompatible with animals with limb deficits22 and, as such, hampers our capacity to investigate depression-like behaviours in rodents.\n\nTherefore, the aim of this study was to investigate the effect of stroke on some very basic tests of exploration, motivation and escape behaviours. We used the distal permanent model of MCAO (pMCAO), which gives consistent cortical lesions with minimal surgical trauma, and aimed to study behaviour and lesion volume to determine whether this model induced a depression-like phenotype.\n\n\nMethods\n\nAnimals: Adult (8–10 weeks), male C57BL/6/J mice (Taconic Ltd., Ry, Denmark) were housed in individually ventilated cages with a standard sawdust and nesting material mix, and kept under diurnal lightning conditions with ad libitum access to food and water. Animals were housed in groups of 5 and an n of 5 was used per group, a single animal was considered as one unit. Animals were pre-assigned to time and sham/stroke groups (see Animal Numbers, Dataset 123). Experiments were approved by The Danish Animal Inspectorate under the Ministry of Food and Agriculture (permit number: 2013-15-2934-00924) and all efforts were made to minimize suffering, distress and lasting harm.\n\nSurgery: All surgeries were performed under isoflurane anaesthesia (2% in O2). The distal part of the left middle cerebral artery (MCA) was permanently occluded as previously described24,25. Briefly, an incision was made between the lateral part of the orbit and the external auditory meatus. A burr hole was made in the skull above the distal part of the MCA. The artery was occluded using bipolar forceps coupled to an electrosurgical unit (ICC 50, Erbe) causing local vessel coagulation, and ensuring a restricted cortical infarct. Animals received a local infusion of lidocaine at the injury site as well as subcutaneous saline/Temgesic and were allowed to recover in a heated environment for 30 minutes prior to being returned to normal housing conditions. No adverse events occurred during surgery.\n\nTissue collection and immunohistochemistry: Animals were surgically anaesthetized using pentobarbital (200 mg/ml) containing lidocaine (20 mg/ml; Glostrup Apotek, Denmark) and transcardially perfused with ice-cold 0.9% saline, followed by 4% paraformaldehyde. Brain tissue was cryoprotected in 30% sucrose and cut at 10µm in a coronal orientation. Individual series were stained with cresyl violet and infarct volumes were analysed using Aperio ImageScope (Aperio CS2 Scanner, Leica Biosystems, UK).\n\nBehavioural tests: All behavioural tests were performed during the light phase following the NC3Rs guidelines and we have used the ARRIVE checklist when writing our report26. Behavioural analysis was videotaped and analysed blind to surgery.\n\nOpen field: This test was performed as previously described27 in a 45cm3 box with observations taking place over a 10 minute period. Movement was tracked using automated software (SMART v3.0, Panlab, Spain) connected to a camera positioned above the box. The area is divided into peripheral and central units, and locomotion and rearing can be recorded in these units. In the open field, low levels of engagement and interaction with the environment (staying close to the wall, moving around little) are traditionally thought to be traits describing emotionality28. Locomotion, rearing and time spent in certain predefined areas of the open field can be measured automatically, rearing was measured manually.\n\nMarble burying: This test was performed as previously described29 in an open Perspex mouse home-cage (44 × 28 × 12cm) with 5cm of sawdust bedding. Twenty marbles were placed at 2cm intervals in 5 lines of 4. Animals were allowed to explore the cage for 30 minutes and the latency to start digging behaviours and the total number of marbles buried to at least 2/3 of their surface was counted. Latency was analysed manually post-hoc from video footage.\n\nTail suspension: Animals were individually suspended for a period of 6 minutes. Movement was analysed manually post-hoc from video footage. Immobility was defined as a period of no less than 3 seconds where no active movement was observed, escape activity was measured using a stop-watch and subtracted from the total time measured (6 minutes). Latency to the first period of consistent immobility (≥3 seconds) was recorded on a second stop-watch.\n\nSucrose preference: This test was performed as previously described30. Animals were acclimatized to sucrose with a 1% solution for 24 hours 7 days prior to surgery. No food or water deprivation was implemented. Baseline testing occurred the day prior to surgery. Mice were given 12 hours of free choice between either 1% sucrose or normal drinking water during the dark (active) phase of their cycle. This time frame excluded the possibility of using this test at the 6 hour survival time. The position of the solutions in the cage was switched at 6 hours to prevent side-bias. Percentage preference for sucrose was calculated at the end of the test using the following formula: Sucrose Preference = VolumeSucrose solution/(VolumeSucrose solution + VolumeWater) × 100.\n\nStatistical analysis: All data were analyzed using Graphpad Prism software (version 7; GraphPad Prism Inc., La Jolla, CA). Analysis of data was performed using two-way analysis of variance (ANOVA) or one-way ANOVA, as appropriate. Data were considered significant at p<0.05. Tukey's multiple comparisons post-hoc testing was applied as appropriate. All data sets are presented as mean ± standard error of the mean (SEM); n are included in figure legends.\n\n\nResults\n\nIn order to carry out basic characterization of the distal pMCAO model over a short time, we took brains at 6 hours, 24 hours, 2 days, 5 days and 7 days post-surgery. At no time point did sham animals show any significant CNS histopathology. Distal pMCAO animals showed a variation in infarct volumes over the time course, peaking at 24 hours (Figure 1A) with the infarct being restricted to the cortex at all time points. However, overall the ANOVA showed no statistical significance. Similarly weight loss in pMCAO animals was higher overall than sham animals (Figure 1B; two-way ANOVA; p<0.05) but no single time-point was significantly different from its sham counterpart.\n\nAnimals underwent permanent occlusion of the distal middle cerebral artery and were allowed to survive for 6 hours, 24 hours, 2 days, 5 days and 7 days. Sham animals received surgery only with no occlusion of the artery. (A) Infarct volumes (mm3) in animals after surgery, sham animals showed no lesions. (B) Weight loss after pMCAO and sham surgeries. Data are presented as mean ±SEM, n=5.\n\nAnalysis of behaviour in the open field test (OFT) results in a number of useful metrics. Distance and speed demonstrate the capability for exploration, rearing behaviours show the motivation for exploration, and measuring the amount of time spent in the central zone vs the perimeter, shows the degree of anxiety the animal might be experiencing. Original studies of open field behaviour suggested that ‘emotional’ animals showed little interaction or engagement with their environments, indicative of a depression-like phenotype31. In our hands distance, speed and rearing were all generally below the levels shown by totally naïve animals (Figure 2A–C; represented by dotted line). Speed, distance and rearing behaviours were all reduced when pMCAO was taken as the main effect (Figure 2A–C; two-way ANOVA; p<0.05, p<0.05, p<0.01, respectively). In the majority of tests, no difference was found at any single time-point between pMCAO animals and their sham counterparts in post-hoc analyses. The exception being at 24 hours where rearing behaviour was significantly decreased compared to sham (Figure 2C; Tukey’s post-hoc test; p<0.05). At no point did animals change the amount of time they spent in the central zone vs the peripheral zone (Figure 2D).\n\nAnimals underwent permanent occlusion of the distal middle cerebral artery and were allowed to survive for 6 hours, 24 hours, 2 days, 5 days and 7 days. Sham animals received surgery only with no occlusion of the artery. (A) Distance travelled in the open field (cm). (B) Speed travelled in the open field (cm/s). (C) Number of rears observed during the period of open field testing. (D) Ratio of time spent in the centre:perimeter areas of the open field. (E) Latency to begin digging in the marble burying test (sec). (F) Total number of marbles buried to at least 2/3 of their surface. Data are presented as mean ±SEM, n=5, dotted line represents naïve animals.\n\nThe digging behaviour seen in the marble burying test has been shown to be a motivational need in laboratory mice, rather than a direct measure of anxiety32,33. Here, all animals showed similar levels of motivation to dig the substrate material when compared to naïve animals, as measured by the latency to start digging (Figure 2E) and the total number of marbles covered in substrate (Figure 2F). At no point during the test were sham and pMCAO animals significantly different from each other.\n\nThe tail suspension test (TST) is a commonly used test to explore motivational escape-like behaviours in the study of depression34. Similarly, a lack of sucrose preference has been shown to be a good indicator of anhedonia, a depression-like sign in rodents21. In both the TST and sucrose preference tests there was no overall difference between post-surgery animals and naïve animals in any of the metrics analysed (Figure 3). In the TST, both latency to the first episode of immobility, and total duration of immobility were not affected by pMCAO, when these animals were compared to shams. Sucrose preference also did not decrease after surgery at any time. In order to determine whether preference was simply a facet of hypodipsia, total volume of liquid drunk was also measured. Whilst this was slightly decreased at 24 hours in all animals when compared to naïve, this did not reach significance, and at no time was the total volume of water missing from the bottles different in pMCAO and sham animals.\n\nAnimals underwent permanent occlusion of the distal middle cerebral artery and were allowed to survive for 6 hours, 24 hours, 2 days, 5 days and 7 days. Sham animals received surgery only with no occlusion of the artery. (A) Distance travelled in the open field (cm). (A) Latency to first period of immobility (≥3 seconds) in the tail suspension test. (B) total time spent immobile in the tail suspension test. (C) Percentage sucrose preference as measured over 12 hours. (D) Total fluid intake as measured over 12 hours. Data are presented as mean ±SEM, n=5, dotted line represents naïve animals.\n\n\nDiscussion\n\nThe emotional disturbances associated with post-stroke depression can severely hinder recovery and, as such, are a crucial area for intervention. However, current anti-depressant therapies, even in uncomplicated major depressive disorder, are ineffective in up to 60% of patients35. Investigating new avenues for therapy in rodent models is a key way of addressing this. However, the rodent models used must be representative of the human condition. There is a long-held belief within the pre-clinical stroke community that the distal pMCAO model does not show any behavioural deficits which could constitute a depression-like phenotype but, to date, there are no citeable articles. This is likely due to a general publication bias in favour of positive data7,8. This study demonstrates that the distal MCAO model of stroke results in cortical lesions but no significant depression-like behaviours.\n\nThe distal pMCAO model produces a robust cortical lesion which varies slightly over time. This data is in line with others showing a large lesion at 24 hours22,25. This reduction in the manifest infarct is due to lesion resolution and the destruction of the core of dead tissue, largely by microglia and infiltrating macrophages36. The location of the lesion potentially contributes to the development, or lack thereof, of post-stroke depression, although results from studies thus far remain controversial37. In humans, there is some evidence for hemispheric bias but these results seem to be hampered by poor patient recruitment, inconsistent methodology and poor controls. Left-hemisphere lesions have been shown to be more commonly correlated with depressive symptoms than right hemisphere lesions38,39. However, a systematic review has shown that right-sided lesions may contribute in the sub-acute phase40. Whilst some degree of lateralization exists in the rodent brain41 the degree to which this contributes to emotionality, is still under scrutiny. Rather, the location of the lesion within the cortex may be the cause for the lack of an affective phenotype. Studies using the focal endothelin-1 (ET-1) model of stroke, where the ET-1 was introduced into the pre-frontal cortex, have shown depressive-like behaviours at the chronic time point (>1 week)42. The pre-frontal cortex is known to play a major role in depression in humans43,44 and whilst its existence in rodents is controversial45, the shift in location from the more functional areas of the cortex - such as the motor and sensory areas, to the PFC - may be the reason for the depression-like behaviours in the ET-1 model used by Vahid-Ansari and colleagues.\n\nEmotionality in rodents has been traditionally challenging to study. Indeed, in models of depression controlling for slight variables in testing regime can significantly affect outcome21. This model of stroke has been repeatedly shown to have minor functional deficits in forepaw function20,22. In models of stroke, there has to be some consideration given to a lower degree of locomotor capacity. For example, the tail suspension test remains more appropriate than the forced swim test because of potential motor deficits shown by the MCAO animals. In this study, our data show that there is no significant change in anhedonic behaviours, either tail suspension or sucrose preference, even at 7 days post-stroke. In a bilateral model of global ischemia in the mouse, depression-like behaviours – as measured by sucrose preference and tail suspension - were not present at 7 days, but did develop at one month post-stroke46. Liu and colleagues suggest that neuronal loss within the hippocampus and hypothalamus may be responsible for this change in behaviour, and that these manifest at later time points in their model. It is possible that there could be some degree of persistent inflammation which continues after 7 days20, which may result in the development of depressive-like behaviours, but this was beyond the scope of this study.\n\nIn conclusion, this study demonstrates that whilst the distal permanent MCAO model produces a robust cortical lesion and is known to demonstrate markers of the ischemic cascade, relevant to interventional studies47, up to 7 days there are no significant affective components to this model and as such, those wishing to investigate depression-like behaviours in rodents post-stroke should consider using alternate models. Whilst this study may have been slightly underpowered in terms of animal numbers, actively pursuing significance with higher numbers would be ethically questionable, and against the principles of reduction and refinement. This data provides citeable evidence that there is no overt depression-like phenotype in the acute phase of the distal MCAO model.\n\n\nData availability\n\nDataset 1: Zip file containing all underlying behavioural and lesion volume data 10.5256/f1000research.15769.d21700223", "appendix": "Grant information\n\nThis work was funded by the Carlsberg Foundation [2012_01_0125 to YC and 2007_01_0176 to KLL].\n\n\nAcknowledgements\n\nThe authors acknowledge the technical assistance provided by Louise Lykkemark and Dorte Lyholmer.\n\n\nReferences\n\nSrivastava A, Taly AB, Gupta A, et al.: Post-stroke depression: prevalence and relationship with disability in chronic stroke survivors. Ann Indian Acad Neurol. 2010; 13(2): 123–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarker-Collo SL: Depression and anxiety 3 months post stroke: prevalence and correlates. Arch Clin Neuropsychol. 2007; 22(4): 519–31. 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PubMed Abstract \| Publisher Full Text\n\nSherwin C, Haug E, Terkelsen N, et al.: Studies on the motivation for burrowing by laboratory mice. Appl Anim Behav Sci. 2004; 88(3–4): 343–358. Publisher Full Text\n\nThomas A, Burant A, Bui N, et al.: Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology (Berl). 2009; 204(2): 361–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCan A, Dao DT, Terrillion CE, et al.: The tail suspension test. J Vis Exp. 2012; (59): e3769. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKirsch I, Moore TJ, Scoboria A, et al.: The emperor's new drugs: An analysis of antidepressant medication data submitted to the U.S. Food and Drug Administration. Prev Treat. 2002; 5(1): 23a. Publisher Full Text\n\nClark RK, Lee EV, Fish CJ, et al.: Development of tissue damage, inflammation and resolution following stroke: an immunohistochemical and quantitative planimetric study. Brain Res Bull. 1993; 31(5): 565–72. PubMed Abstract \| Publisher Full Text\n\nNickel A, Thomalla G: Post-Stroke Depression: Impact of Lesion Location and Methodological Limitations-A Topical Review. Front Neurol. 2017; 8: 498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajashekaran P, Pai K, Thunga R, et al.: Post-stroke depression and lesion location: A hospital based cross-sectional study. Indian J Psychiatry. 2013; 55(4): 343–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHackett ML, Yapa C, Parag V, et al.: Frequency of depression after stroke: a systematic review of observational studies. Stroke. 2005; 36(6): 1330–40. PubMed Abstract \| Publisher Full Text\n\nWei N, Yong W, Li X, et al.: Post-stroke depression and lesion location: a systematic review. J Neurol. 2015; 262(1): 81–90. PubMed Abstract \| Publisher Full Text\n\nKim S, Mátyás F, Lee S, et al.: Lateralization of observational fear learning at the cortical but not thalamic level in mice. Proc Natl Acad Sci U S A. 2012; 109(38): 15497–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVahid-Ansari F, Lagace DC, Albert PR: Persistent post-stroke depression in mice following unilateral medial prefrontal cortical stroke. Transl Psychiatry. 2016; 6(8): e863. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer JH, Kapur S, Houle S, et al.: Prefrontal cortex 5-HT2 receptors in depression: an [18F]setoperone PET imaging study. Am J Psychiatry. 1999; 156(7): 1029–34. PubMed Abstract\n\nBeauregard M, Leroux JM, Bergman S, et al.: The functional neuroanatomy of major depression: an fMRI study using an emotional activation paradigm. Neuroreport. 1998; 9(14): 3253–8. PubMed Abstract \| Publisher Full Text\n\nUylings HB, Groenewegen HJ, Kolb B: Do rats have a prefrontal cortex? Behav Brain Res. 2003; 146(1–2): 3–17. PubMed Abstract \| Publisher Full Text\n\nLiu S, Han S, Dai Q, et al.: BICAO-induced ischaemia caused depressive-like behaviours and caspase-8/-9-dependent brain regional neural cell apoptosis in mice. Stroke Vasc Neurol. 2017; 3(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLambertsen KL, Biber K, Finsen B: Inflammatory cytokines in experimental and human stroke. J Cereb Blood Flow Metab. 2012; 32(9): 1677–98. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "38091", "date": "18 Sep 2018", "name": "Ulrich Dirnagl", "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\nGammelstrup Andersen and collegues describe experiments in a murine stroke model to investigate whether post-stroke depression (PSD), a frequent complicaton of human stroke, can be modeled experimentally. In sample sizes of 5 per group, using young male C57/Bl6/J mice, a left sided permanent occlusion of the distal middle cerebral artery (which produces neocortical infarcts) they conducted behavioural tests focusing ond exploration, motivation, as well as hedonia and despair (which are considered as proxies for depression by some). Despite all animals demonstrating reproducible infarcts, no ‘depression like’ phenotype was observed in this model. Although the methodology is sound in principle, and the research question relevant, there are a number of issues which negatively affect the impact of this work in its present form.\nGroup sizes of 5 are exceedingly low, but the sentence ‘Whilst this study may have been slightly underpowered in terms of animal numbers, actively pursuing significance with higher numbers would be ethically questionable, and against the principles of reduction and refinement.’ is absurd and unscientific. In fact, underpowered studies are unethical, as pointed out by Cressey D1. An informal, post hoc power calculation on their data set reveals that they may have achieved only 10-20 % power (instead of 80 or 95%). This is particularly worrisome, as they report a NULL result, so false negatives are a major concern. The study is lacking a justification of the sample sizes, and Type II error considerations. What are the effect sizes the study could have possibly detected? In general, the test statistics of this study don’t make a lot of sense. Although they are formally correct, in the absence of the definition of one primary outcome, and the fact that they performed at least 12 ANOVAs (for which they did not correct), as well as given the exploratory character of the study, test statistics should be avoided, and the focus should be on effect sizes and variance. This brings me to another problem: The authors use SEMs (a measure of precision, not of the spread of the data) and they use bar graphs, instead of dot plots (individual data points) and true measures of variance to illustrate their data. The authors do not mention how many animals went into the study (ARRIVE guidelines), they only mention group sizes (in the figures). Only downloading the full data set reveals that there obviously was no attrition, although there are unexplained missing data points. The authors are strongly advised to consult a statistician. External validity I: Left sided occlusion only! Kronenberg et al2, in a mouse model of transient proximal occlusion demonstrated that left, but not right, middle cerebral artery occlusion leads to chronic ‘Depression-Like’ behavior, which was reversed by serotonin reuptake inhibition . This article, as well as a number of other relevant publications pertinent to the subject of modeling of PSD in rodents, is not cited. Although Gammelstrup Andersen used left sided occlusion, it at least demonstrates the potential of lateralization of behavioural symptoms in rodents. External validity II: Only male mice are studied, and of an age range and without comorbidities. This does not reflect the cohort of patients which are at risk for stroke and PSD. The title of the study ‘Distal middle cerebral artery occlusion does not result in depression-like behaviors’ is imprecise and should contain at least the species, but ideally more information on the model. I suggest: An exploratory investigation of ‘depression-like’ behaviours in a model of left-sided distal middle cerebral artery occlusion in young, male C57B6 mice.\n\nIn summary, I strongly recommend to change the graphical presentation of the data, redo the statistics (descriptive instead of test), discuss the limitations (in particular the exceedingly low power, and issues of external validity), and include some of the missing literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "40488", "date": "27 Nov 2018", "name": "Paul Albert", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript examines male C57BL6 mice with thermocoagulation of the distal left middle cerebral artery occluded.\n\nOverall the study has several flaws. First the cohort size (5/group) is insufficient and this needs more replication (n = 10-15/group is typical, but larger may be necessary due to stroke variability) to detect behavioral changes. Second, the lesion site is not sufficiently characterized. Third, control measures of fine locomoter activity are not done. Fourth, the tests used are not effective in the strain or not appropriate to assess anxiety/depression. Fifth, only one test of depression test is done; ideally, 2-3 tests for each phenotype need to be done.\n\nSpecific comments:\n\nOne confound is whether multiple tests were done in the same day; this needs to be clarified: how many animals were used, what tests were done on each group, how many tests/day on each mouse? Fig. 1A: It would be important to show the size and location of the stroke at 24 hr. These strokes are notoriously variable, hence mapping the stroke is important. More details and quantification of the location (Bregma) of the incision and stroke need to be provided. Since the stroke is so large and variable (esp. at 24 h), additional studies to assess fine motor impairments are critical (e.g., ladder test, cylinder test, etc. Motor impairments can affect results in locomotor, anxiety and depression tests. Fig. 2: At none of the time points was any of the outcomes significant, only an overall significance. More data points are needed to test when there is impairment or recovery. Rearing is not specific measure for anxiety: validated tests need to be done (e.g., elevated plus maze, centre time in open field, etc.). Fig. 2: Marble burying was also maximal 20 marbles for all groups, thus may be to insensitive to detect motor impairments. Fig 3. In the sucrose preference test as performed, almost 100% of the mice show 100% preference for sucrose. This raises concern about the sensitivity of the test to detect anhedonia in C57BL6 mice: this strain of mice is unresponsive in this assay even after 9 weeks of chronic stress (Pothion, 2004). Hence the data are inconclusive. Fig. 3: The data for TS are not convincing; the error seems too high since N value is too low. The dashed line needs to show N, mean ± SE of naïve mice: why are sham having more immobility than naïve?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-1430
https://f1000research.com/articles/7-723/v1	11 Jun 18	{ "type": "Research Article", "title": "Areca nut extract demonstrated apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma", "authors": [ "Liza Meutia Sari", "Gus Permana Subita", "Elza Ibrahim Auerkari", "Gus Permana Subita" ], "abstract": "Background: Oral squamous cell carcinoma is a neoplasm of keratinocyte cells of oral mucosa epithelium that can potentially spread through lymphatic tissue or blood vessel. Although areca nut is one of the plants with risk of inducing that cancer, areca nut is believed to have high antioxidant properties. Due to the current interest in the apoptosis effects from areca nut for oral cancer treatment, we investigated its ability to induce apoptosis and caspase-3 activity in oral cancer cell lines: HSC-2 and HSC-3. Methods: We examined the effect of areca nut on apoptosis and caspase-3 activity in HSC-2 and HSC-3 cells. Flow cytometry was conducted for the quantification of the cells that were apoptotic and expressing the caspase-3 enzyme for 24 and 48 hours. Results: Areca nut induced a significant increase (p<0.01) in late apoptosis of HSC-2 cells and mostly occurred over 48 hours. The study also found that in HSC-3, there were significant increases (p<0.01) the percentage of cells in early apoptosis after 24 hours and late apoptosis at 48 hours. Caspase-3 activity increased after 24 and 48 hours of areca nut exposure in both cells. Conclusions: The study showed that areca nut could be considered as a potential anticancer agent through its capability in inducing a caspase-dependent apoptosis.", "keywords": [ "Areca nut", "oral cancer", "apoptosis", "caspase-3" ], "content": "Abbreviations\n\nDNA, Deoxyribose Nucleic Acid; AIF, Apoptotic Inducing factor; AP-1, Activator Protein-1; Bcl-2, B-cell lymphoma-2; COX-2, Cyclooxigenase-2; DISC, Death Inducing Signal Complex; EGF, Epidermal Growth Factor; FDA, Food and Drug Administration; FITC, Fluorescein Isothiocyanate; IC50, Inhibition Concentration 50; IGF-1, Insulin Growth Factor-1; MAPKs, Mitogen Activated Protein Kinases; PI, Propidium Iodide; WHO, World Health Organization.\n\n\nBackground\n\nCancer originates from a multistep process which is modulated by environmental and genetic factors1. Cancer cells undergo pathologic proliferation and no longer respond to expression signals from tumor suppressor genes, causing disruption of cell cycle phases which acts to repair DNA, and eventually become antiapoptotic cells1,2. Cell cycle inhibition and apoptosis induction are two strategies in treating cancer which are considered forms of targeted therapy3. Cancer cells lose the ability to control these two mechanisms4. The ability of a anti-neoplastic drug is to induce cell cycle inhibition and apoptosis highly influences its potency as a cytotoxic agent. An effective chemopreventive agent should preferably interfere early in the process of carcinogenesis to eliminate premalignant cells before they acquire malignant character5. Apoptosis is the process of programmed cell death, and is dependent on cysteine protease enzymes called caspases6,7. There are two pathways involved in the initiation of apoptosis, the intrinsic and extrinsic pathway6,7. These two pathways ultimately lead to the activation of executioner caspases, caspases 3, 6, and 77. Expression of caspase-3 is significantly lower in tumor tissue compared with normal tissue and tissue surrounding the tumor8. The caspase-3 is a key effector caspase in the apoptotic program of cell suicide. The lack of caspase-3 expression may lead survival of cancer cell so that it will increase the severity of cancer.\n\nNatural compounds are important in the treatment of life threatening conditions. In many surveys, herbal medicines are amongst the most commonly used group of treatment. Herbal remedies are believed by the general public to be safe, cause less side effects and less likely to cause dependency. According to the World Health Organization (WHO), poverty and poor access to treatment cause approximately 65%–80% of world population living in developing countries to still depend on natural ingredients of plants for medicine as they are much more affordable9. Development of herbal drugs in the internationally has increased rapidly, with China, Europe and United States as the largest suppliers. The percentage of herbal drug users has reached 90% in Ethiopia, 70% in India and Chile, and 40% in China and Colombia10. One study found that four in ten adults in the United States currently uses traditional alternative treatment11. 60% of the drugs approved by the US Food and Drug Administration (FDA) since 1984–1994 are isolates from plants12. Of the 121 types of drugs prescribed for cancer treatment, 90 are derived from medicinal plants10.\n\nOne study reports that of the 65 new drugs listed for cancer treatment since 1981–2002, 48 originated from natural products derived from plants13. Research and development of herbal medicines is needed to produce drugs which can be approved by formal health care agencies, especially in terms of their quality, safety and efficacy14.\n\nOne of the plants with potential to be developed as a herbal medicine is the pinang plant (Areca catechu Linn; areca, Palmaceae). Indians and Malaysians chew this seed to refresh breath, smooth digestion, increase sexual desire, eradicate helminths, and maintain stamina15. Areca nut is believed to be able to induce euphoria, a tranquilized condition, with warm and comforting effects. The activities of areca nut effects include antioxidant and antihelmintic16–24, antidiabetic25, antidepressant20, antifungal24, antibacterial26, antimicrobial27, antimalarial28, anti-inflammatory22, insecticide, psychoactive, hepatoprotective29, and larvicidal30, antiaging and cosmetic31, hypolipideamic32 and hypoglicemic33. Other studies, however, have identified negative effect from excessive areca nut consumption specifically carcinogenic properties which can induce oral squamous cell carcinoma (OSCC)34. The carcinogenic effect of areca nut is caused by nitrosamine that was produced by nitrosation process by alkaloid (arecoline) from dry areca nut when chewed or digested in acidic condition in gastric for long term and uncontrollable35. The incidence of OSCC can also be influenced by several other both intrinsic (abnormalities or mutation of tumor suppressor genes and oncogenes) and extrinsic (smoking tobacco, vitamin A and iron deficiency, candida infection, viral infection, and immunosuppression). Areca nut is traditionally masticated either alone or along with a large variety of ingredients, such as betel leaf (family Piperaceae), Uncaria gambir, and slaked lime for traditional ceremonial cultural roles in Indonesia. However, there are no current reports on the apoptotic mechanism of the areca nut extract on oral squamous cell lines.\n\nHence in this study, the ability of areca nut to induce apoptosis and caspase-3 activity was evaluated and compared between two different time periods (24 and 48 hours) and two types of OSCC cell lines, human squamous carcinoma HSC-2 and HSC-3.\n\n\nMethods\n\nThe study materials were obtained from areca nuts of pinang plant from Aceh Besar, Indonesia, which was determined and documented by the Botanical Division of Biological Research Center LIPI Cibinong, complete with its roots, stems, leaves, flowers, and seeds in 2017.\n\nThe sample used was two kilograms of areca nut (gross weight). Areca nut was collected and cleansed from dirt (wet sortation), then washed with running water until clean and drained. Those seeds were dried in open air and covered from direct sunlight then continued with drying using oven at 50°C. Dried simplicia (unprocessed natural ingredient) was crushed using a blender producing a powdered simplicia and sifted with 20 mesh sieves. The powder was macerated with 96% ethanol solvent. Around 500 grams powdered simplicia was put into container then 1 L of 96% ethanol was added, closed, and left for three days covered from sunlight, while repeatedly stirred. After three days the extract was strained, and the remaining extract then was dried. The dried extract was added to 500 mL of 96% ethanol and stirred, after acquiring all extract. The container was closed, left in a cool place and covered from sunlight for two days. The sediment was separated and liquid extract was obtained. Then the extract was evaporated using rotary evaporator at 30–40°C then concentrated again using water bath so a dense extract of areca nut would be obtained.\n\nThe HSC-2 and HSC-3 cell lines were cultured in complete Dulbecco’s modified Eagle’s medium (D6429, Sigma-Aldrich) containing 10% FBS, nonessential amino acids, pyruvate, glutamine, and vitamins at 37°C with 5% CO2/95% air in a humified CO2 incubator. All media were also supplemented with 100 units/mL of penicillin and 100 mg/mL of streptomycin (15070063, Thermo Fisher Scientific). The above-mentioned cell lines were procured more than 6 months ago and have not been tested recently for authentication in our laboratory. The HSC-3 and HSC-2 cell lines used in this study were provided by the Oral Biological Laboratory, Faculty of Dentistry of the University of Indonesia. The HSC-3 cell line was derived from an oral squamous cell carcinoma of the tongue with a p53 gene mutation, namely a 4bp insertion or change in amino acid in the form of TAAG insertion in codon 305–306, exon 836. The HSC-2 cell line was also derived from an oral squamous cell carcinoma of the tongue but without the p53 gene mutation37. Cell lines, placed in cryophilic liquid N2, were then moved into 15 mL tube, then PBS (10010031, ThermoFisher Scientific) was added up to 10 mL. The thawing process started with centrifuging by using Laboratory benchtop centrifuge Liston C 2201 for 10 min at 300 × g at room temperature, the supernatant was disposed, the cell concentrate at the base of the tube (pellet) was added to 2–3 mL complete DMEM medium, and then it was pipetted to culture a plate containing 7–10 mL DMEM medium and was spread evenly. It was incubated at 37°C with a 5% CO2/95% air in a humified CO2 incubator . Media was changed by removing old medium from the culture plate by pipetting, rinsing with PBS two to three times, pouring new complete DMEM medium (around 7 – 10 mL) and then placing back into the incubator. If the cells achieved 80% confluents, then the confluents was ready to be harvested. The medium was disposed and rinsed with PBS Ca2+ and Mg2+ two to three times with volume of 2 mL, then 1 mL Trypsin EDTA (59418C, Sigma-Aldrich) was added, then it was incubated for five to ten minutes. After addition of complete DMEM (2 – 3 ml) and transferring into a 15 mL tube by pipetting, and centrifuging at 500 rpm for 10 minutes, the supernatant was discarded. The pellet was homogenized by pipetting, and the resuspended cells with the culture medium was ready to be used for experiment and cell counting with a hemocytometer. We had performed the cell viability assay previously to evaluate the percentage cytotoxicity and IC50 of areca nut extract after treating the HSC-2 cells for 72 hours is 629.50 µg/mL while in HSC-3 cells is 164.06 µg/mL38.\n\nThe HSC-2 and HSC-3 cells were plated at 1 × 105 cells/well in 60 mm dishes with DMEM. Areca nut extract (629.50 µg/mL) was added for HSC-2 cells and 164.06 µg/mL for HSC-3 cells. For combination experiments, areca nut extracts were added at the same time and both were incubated for 24 and 48 h, before the preparation of cell extract or quantification of apoptosis and caspase-3 activity (see below).\n\nA flow cytometry was used to analyze tubes containing cells with and without extract material after 24 and 48 hours exposures. Cultures of HSC-2 and HSC-3 cells with 1×105 cells/mL concentration were centrifuged for five minutes with 500 rpm speed, washed with 1 mL cold PBS (10010031, ThermoFisher Scientific), and re-centrifuged for five minutes and vortexed. One hundred µl test solution containing 1x105 cells in each tube is resuspended with binding buffer. 5 µL FITC Annexin V (556547, BD PharmingenTM) and 5 µL PI (556547, BD PharmingenTM) stains were added to these cells and incubated for 15 minutes in a dark place, analyzed by flow cytometry (BD FACS Calibur Flow cytometry System type E 34297502328, .San Jose, California, USA) and by manual gating using CellQuest software (Becton Dickinson, NJ). Gating was performed on blinded samples\n\nCells were collected with and without areca nut extract for 24 and 48 hours, respectively. Prepared HSC-2 and HSC-3 cells (1×105 cells/mL, 5 mL) were washed with cold PBS and resuspended with 400 µL BD Cytofix/CytopermTM Solution (51-6896KC, BD PharmingenTM). The procedure was began by determining the amount of BD Perm/WashTM buffer (51-6897KC, BD PharmingenTM) and 20 µL Rabbit anti-active caspase-3 polyclonal antibody (351-68655X, BD PharmingenTM) required, so that each test was consist of 100 mL BD Perm/WashTM buffer and 20 µL antibody. After incubation for 20 minutes on ice, cells were centrifuged and washed with BD Perm/WashTM buffer. After that, BD Perm/WashTM buffer was added and then the antibody is incubated for thirty minutes in room temperature. Each tube was rinsed again with 1 mL BD Perm/WashTM buffer, re-centrifuged, then added 300 µL BD Perm/WashTM buffer.\n\nAll data were presented as the mean ± standard deviation of triplicate parallel measurements. Statistical analysis used SPSS 10.0 and the data were analyzed with the unpaired t-test using significance level of p<0.01.\n\n\nResults\n\nApoptosis assay was performed on cell population with and without areca nut extract for 24 and 48 hours. The IC50 dose of extract used was 629.5 μg/mL for HSC-2 cells. The percentage value of the cell population count was calculated based on the division of four quadrants, i.e. the viable cells (lower left quadrant; AV-/PI-), early apoptosis (lower right quadrant; AV+/PI-), late apoptosis (upper right quadrant; AV+/PI+), and necrotic cells (upper left quadrant; AV-/PI+). The results of the apoptosis assay in 24 hours showed an increase in percentage cell number after areca nut extract treatment undergoing late apoptosis, as much as 83.82±15.86%. This number is 68.28% higher compared to controls, or approximately 5.4 times higher than control (15.54±23.52%). This increase was significant, suggesting that a reduction of viability represents mostly apoptosis.\n\nThen, we examined the effect of areca nut extract after 48 hours exposure. The result showed that areca nut also induced an increase in late apoptosis cell after 48 hours. As can be seen in Figure 1, late apoptotic cells with pink and red dots in upper right quadrant indicated that areca nut was high cytotoxicity. Therefore, it can be concluded that areca nut extract is capable of inducing apoptosis in HSC-2 cells. Graphs showing a comparison of mean percentage between control cells and after areca nut extract exposure is shown in Figure 1.\n\nA. Flow cytometry analysis for apoptosis inducing activities of areca nut on HSC-2 cells, a and c: control; b and d: treated with areca nut. B. Graph of comparison between percentage of HSC-2 cells with and without 24 and 48 hours extract exposure at IC50.\n\nThe apoptosis assay performed in HSC-3 cells demonstrates a different result to that of HSC-2 cells after areca nut extract exposure for 24 hours. There was no increase in late apoptosis but, instead the early apoptotic cell population increased. There was an increase in early apoptotic cell populations from untreated to treated cells (1.77% to 17.88%, resepectively). The apoptosis assay in HSC-3 cells after 48 hours exposure, however, shows an increase in early and late apoptotic cell percentage.\n\nFigure 2B shows that, in HSC-3 cell lines, areca nut extract induced only early apoptosis after 24 hours, but both early and late apoptosis were markedly enhanced after 48 hours. During apoptosis, cell shrinkage occurs, which is associated with a decrease in forward scatter. Further, the formation of apoptotic vesicles in the cells during apoptosis leads to an increase side scatter profile.\n\nA. Flow cytometry analysis for apoptosis inducing activities of areca nut on HSC-3 cells, a and c: control; b and d: treated with areca nut. B. Graph of comparison between HSC-3 cell percentage with and without 24 and 48 hours areca nut extract exposure at IC50.\n\nCaspase-3 assay was performed in triplicate in HSC-2 cells also using flow cytometry. The value is calculated based on percentage of cell population with caspase-3 enzyme activity during apoptosis. The percentage of control and test cells in the same quadrant was compared. The M1 quadrant is demonstrates the number of living cells without active caspase-3, whereas M2 quadrant is number of apoptotic cells with active caspase-3. Areca nut extract caused an increase in the number of cells with active caspase-3 which is 85.94±56.86% more than the number of cells without activate caspase-3 (14.37±11.27% after 24 hours exposure). This value is in accordance with the results of the apoptosis test, as an increase in caspase-3 corresponds with an increase of late apoptosis cell population. Untreated cells (M1) were primarily negative for the presence of active caspase-3, whereas greater than one third of the treated cells were positive for active caspase-3 staining (M2). The similar patterns were seen in 24 and 48 hours after exposure (Figure 3A). This shows that the ability of the extract to induce apoptosis is increased with longer exposure in HSC-2 cells.\n\nA. Flow cytometry analysis for caspase-3 activity inducing activities of areca nut on HSC-2 cells, a and c: control; b and d: treated with areca nut. B. Graph of comparison between percentage of HSC-2 cells with active caspase 3 with and without areca nut extract exposure after 24 and 48 hours at IC50.\n\nThe high concentration of active caspase-3 activated in HSC-3 cells, which is increasing 126 times higher than control cells after 48 hours exposure (Figure 4). Population distribution is also clearly shown between cells with and without extract exposure.\n\nA. Flow cytometry analysis for caspase-3 activity inducing activities of areca nut on HSC-3 cells, a and c: control; b and d: treated with areca nut. B. Graph of comparison between percentage of HSC-3 cells with active caspase-3 with and without areca nut extract exposure after 24 and 48 hours at IC50..\n\n\nDiscussion\n\nThis study is the first study which clearly reveals the potential cytotoxicity effect and mechanism of action of areca nut in oral squamous cell lines. This study performs apoptosis and caspase-3 activity tests using flow cytometry, with the objective to acknowledge whether the cell death mechanism happens through apoptosis induction by areca nut extract or not. In order to acknowledge the optimum time of areca nut extract activity against the cells, two units of time are used, which are 24 and 48 hours. The results of flow cytometry analysis on HSC-2 cells shows that areca nut extract can induce late apoptosis activity after 24 and 48 hours exposure, but the increase of late apoptotic cells occurs more following 48 hours exposure. This result is in accordance with past study that performed apoptosis test using orange acridine-ethidium bromide staining (double staining). The result showed that treatment with ethanolic extract of areca nut (IC50 77 μg/mL) for 48 hours inhibits growth of MCF-7 cells as much as 13–84%39.\n\nAreca nut extract can possibly induce non-apoptotic cell death or necrosis. This is shown from the increase in necrotic cell percentage significantly after 24 hours exposure. One of the past studies using catechin from green tea, proved that catechin has the ability to induce necrosis or non-apoptotic cell death in leukemia cells without caspase-8, 9, and 3 activities40 Although molecular mechanism pathway of necrosis is not clearly understood, catechin can possibly induce necrosis through two pathways, which are decreasing concentration of intracellular ATP and interaction on ATP-binding site of glucose regulated protein (GRP78) leading to increased activity of ATPase40. This result shows two competitive abilities between catechin and ATP-binding site leading to necrosis with catechin activity via the apoptosome (intrinsic pathway) and death induced signaling pathway (DISC; extrinsic pathway).\n\nAnalysis of caspase-3 activity in HSC-2 cells shows results in accordance with the apoptosis assay, in that caspase-3 activity increases significantly after areca nut extract for 24 and 48 hours compared to control, with the increase of caspase-3 activity also being higher after 48 hours exposure. This result is similar to the study using catechin of green tea and hydrate catechin against HS-sultan and RPMI8226 cell strains, and MCF-7 cells using Western blot and quantitative RT-PCR techniques, that this compound can induce caspase-3, 8, and 9 activities41.\n\nThe results of flow cytometry analysis on HSC-3 cells, show that areca nut extract could also induce apoptosis after extract exposure for 24 and 48 hours. Unlike with HSC-2 cells, extract exposure induced more early apoptosis after 24 hours exposure, but after 48 exposure apoptosis induction by the extract happened more in the end step. Caspase-3 activity as an effector caspase is shown to be related with late apoptosis activity because of the increase of caspase-3 with increasing late apoptotic cells percentage. There is a significant increase in necrotic cells percentage after 48 hours exposure. Therefore, the apoptosis assay showed that areca nut extract is capable of inducing apoptosis in HSC-2 and HSC-3 cells with an optimum time after 48 hours exposure. Although the extract has the same optimum time in both cells, there is a difference on extract effect on the number of apoptotic cells, where the percentage of HSC-2 cells undergoing apoptosis is higher than HSC-3 cells. This result is possibly because there is a difference of cell sensitivity against areca nut extract. Literature shows that in addition to the p53 gene mutation in HSC-3, severe damage to phosphorylation of Ser46 in HSC-3 cells causes loss of apoptotic ability mediated by p53 and also increased survival ability of HSC-3 cells against anticancer genes compared to HSC-2 cells42. The p53 tumor suppressor gene holds an important role in deciding the cells’ fate if there is DNA damage. If there is mild damage, p53 will stop the growth until the DNA repair process is done. If there is severe damage, p53 will induce senescence process to prevent the increase of precancerous cells. Phosphorylation of Ser46 causes -p53 to activate proapoptotic genes, leading to the induction of apoptosis42. However, the different response between HSC-2 and HSC-3 cells after exposure to extract could possibly be caused by different effects of areca nut extract on the extrinsic and intrinsic pathways. Due to the effects of the p53 mutation on the intrinsic pathway in HSC-3 cells raises the possibility of the extracts effects being solely through the extrinsic pathway, whereas in HSC-2 cells, the extract works on the extrinsic and intrinsic pathways leading to more apoptosis occurring in HSC-2 cells. This cannot be determined from these results as not tests on caspase 8 and 9 were performed.\n\nThe induction of apoptosis in tumor cells is considered a valuable method to treat cancer. A wide variety of natural substances have been recognized to have the ability to induce apoptosis in various tumor cells. Apoptosis is an active form of cell suicide controlled by a network of genes, in which the Bcl-2 family proteins play an important role in control of apoptosis. The balance of pro- and anti-apoptotic Bcl-2 family proteins controls permeabilization of the outer mitochondrial membrane and release of intermembrane space proteins, most notably cytochrome c. In the presence of cytochrome c and dATP, Apaf-1, the scaffold around which the apoptosome is built, recruits and activates caspase-9, which then propagates a cascade of further caspase activation events downstream.\n\nCaspases inside cells are in an inactive form (procaspase), but activation induces the production of other caspases leading to cell death through proteolytic activity43,44. Initiator caspase activation (caspase-8 and 9) by catechin shows early apoptotic activity in cell death. Cell death through extrinsic pathway can be influenced by catechin derivates originating from green tea, resulting in inhibition NF-kB, MAPKs signals, nitric oxide synthesis, and EGFR mediated by transduction pathway signaling through suppressing on EGF binding with its receptor, AP-1, IGF-1 signaling pathway, COX-2, and proteasome activity43. Cell death through the mitochondrial pathway can also be induced by catechin. Changes in mitochondria caused by an increase in membrane permeability leads to opened pores and loss of the mitochondrial transmembrane potential causing release of cytochrome c into cytosol, thereby activating the caspase-9 and 3 pathway43.\n\nCatechin can increase apoptogenic protein release from mitochondria such as cytochrome c, Smac/DIABLO, and AIF into cytosol leading to death signaling from inside of the mitochondria releasing more and activating caspase-345. Decrease of Bcl2 and Bcl-XL antiapoptotic protein, increase of Bax proapoptotic protein in the intrinsic pathway are also influenced by catechin. If there is p53 mutation, the function of BH-3 proapoptotic protein will be inhibited and function of Bcl-2 antiapoptotic protein family will increase leading to inhibition of anticancer agent activity in intrinsic pathway. Caspase-3 activation is a crucial component in the apoptotic signaling cascade. Based on the results obtained from our study, the apoptosis pathway involved in areca nut-induced cell death in both cancer cell lines may be through the extrinsic and intrinsic pathways. Further investigation is needed to clarify the exact mechanism through which areca nut induces apoptosis.\n\n\nConclusion\n\nApoptosis is the main cell death mechanism in HSC-2 and HSC-3 cells after areca nut extract exposure for 24 and 48 hours. This is shown by the high population of early and late apoptotic cells in HSC-2 and HSC-3 cells compared to cells without extract exposure. The optimum time of apoptosis occurrence after areca nut extract is 48 hours. We postulated that one of the possible action for the apoptosis effects of this extract occurred through increased activities of caspase-3 enzyme. This is indicated by the high activity of caspase-3 in HSC-2 and HSC-3 cells compared to cells without extract exposure, which also proves that cell death that happened was late apoptosis. There is great potential to develop areca nut as an adjuvant therapy as a chemotherapeutic agents for oral squamous cell carcinoma treatment, hence additional studies are needed, particularly in vivo studies to further evaluate the observed effect.\n\n\nData availability\n\nDataset 1: Output flow cytometry files for all experiments with statistical analysis output files 10.5256/f1000research.14856.d20633846.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNeville BW, Damm DD, Allen CM, et al.: Squamous cell carcinoma in Oral and maxillofacial pathology. 2nd ed. Philadelphia, Pennsylvania: Saunders; 2002.\n\nChoi S, Myers JN: Molecular Pathogenesis of Oral Squamous Cell Carcinoma: Implications for Therapy. J Dent Res. 2008; 87(1): 14–32. 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International Conference on the Role of Universities in Hands-On Education Rajamangala University of Technology Lanna, Thailand. 2009; 209–15. Reference Source\n\nJiang JH, Jung SY, Kim YC, et al.: Antimalarial effects of Areca catechu L. Korean J Orient Physiol Pathol. 2009; 23(2): 494–98. Reference Source\n\nPithayanukul P, Nithitanakool S, Bavovada R: Hepatoprotective potential of extracts from seeds of Areca catechu and nutgalls of Quercus infectoria. Molecules. 2009; 14(12): 4987–5000. PubMed Abstract \| Publisher Full Text\n\nAmudhan MS, Begum VH, Hebbar KB: A review on phytochemical and pharmacological potential of Areca catechu L. seed. IJPSR. 2012; 3(11): 4151–57. Publisher Full Text\n\nLee KK, Choi JD: The effects of areca catechu L extract on anti-aging. Int J Cosmet Sci. 1999; 21(4): 285–95. PubMed Abstract \| Publisher Full Text\n\nByun SJ, Kim HS, Jeon SM, et al.: Supplementation of Areca catechu L. extract alters triglyceride absorption and cholesterol metabolism in rats. Ann Nutr Metab. 2001; 45(6): 279–84. PubMed Abstract \| Publisher Full Text\n\nChempakam B: Hypoglycaemic activity of arecoline in betel nut Areca catechu L. Indian J Exp Biol. 1993; 31(5): 474–75. PubMed Abstract\n\nZain RB: Cultural and dietary risk factors of oral cancer and precancer--a brief overview. Oral Oncol. 2001; 37(3): 205–10. PubMed Abstract \| Publisher Full Text\n\nIARC Working Group on the Evaluation of Carcinogenic Risks to Humans: Betel-quid and areca-nut chewing and some areca-nut derived nitrosamines. IARC Monogr Eval Carcinog Risks Hum. 2004; 85: 1–334. PubMed Abstract \| Free Full Text\n\nKamiya Y, Ohshima T: The Individual Cell Properties of Oral Squamous Cell Carcinoma and p53 Tumor Suppressor Gene Mutation. Oral Sci Intl. 2005; 2(2): 104–17. Publisher Full Text\n\nOkamura M, Shimada J, Sakagami H: Comparative analysis of cell death induction by cisplatin and 5-FU in human oral squamous and hepatocellular carcinoma cell lines. Anticancer Res. 2008; 28(1A): 253–9. PubMed Abstract\n\nSari LM, Subita GP, Auerkari EI: Potential antioxidant and cytotoxic activity of areca nut (Areca catechu Linn.) extract in human oral squamous cell carcinoma and keratinocyte cells. Asian J Pharm Clin Res. 2017; 10(10): 286–91. Publisher Full Text\n\nMeiyanto E, Handayani S, Susidarti RA, et al.: Chloroform fraction of Areca catechu L. induces apoptosis and decreases Bcl2 expression on MCF-7 cells. Artocarpus. 2008; 8(2): 55–69.\n\nIwasaki R, Ito K, Ishida T, et al.: Catechin, green tea component, causes caspase-independent necrosis-like cell death in chronic myelogenous leukemia. Cancer Sci. 2009; 100(2): 349–56. PubMed Abstract \| Publisher Full Text\n\nAlshatwi AA: Catechin hydrate suppresses MCF-7 proliferation through TP53/Caspase-mediated apoptosis. J Exp Clin Canc Res. 2010; 29(1): 167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIchwan SJ, Yamada S, Sumrejkanchanakij P, et al.: Defect in serine 46 phosphorylation of p53 contributes to acquisition of p53 resistance in oral squamous cell carcinoma cells. Oncogene. 2006; 25(8): 1216–24. PubMed Abstract \| Publisher Full Text\n\nParrish AB, Freel CD, Kornbluth S: Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol. 2013; 5(6): pii: a008672. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHashimoto T, Kikkawa U, Kamada S: Contribution of caspase(s) to the cell cycle regulation at mitotic phase. PLoS One. 2011; 6(3): e18449. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenturk S, Mumcuoglu M, Gursoy-Yuzugullu O, et al.: Transforming growth factor-beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth. Hepatology. 2010; 52(3): 966–74. PubMed Abstract \| Publisher Full Text\n\nSari LM, Subita GP, Auerkari EI: Dataset 1 in: Areca nut extract demonstrated apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma. F1000Research. 2018. Data Source" }	[ { "id": "36771", "date": "20 Aug 2018", "name": "Irna Sufiawati", "expertise": [ "Reviewer Expertise Oral Medicine", "Immunology", "Microbiology", "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\nThe manuscript is well written and interesting to read. While the findings are not entirely new, they warrant continued attention because of the current interest in the apoptosis effects of areca nut for oral cancer treatment. However, I see the following issues that should be clarified/resolved before indexing this paper:\n1. Ethic statements. It is unclear whether the cell line has been authenticated, and the methods make no statement that an ethics committee or institutional review board approved the study, which involves the use of human cell lines. If the authors received ethical approval, please include the name of the ethics committee and the approval number. 2. In the results section, the figures should be made clearer using the appropriate graphing software. 3. The discussion section needs to be elaborated. While the discussion includes references to the previous studies, it has not been discussed whether the findings of the study corroborate or contradict those of similar previous studies. In addition, it would be better if the authors discuss the important signaling elements MAPK's and NFκB signaling pathways related to areca nut extract in cancer cells. The discussion appears to be redundant with the results and conclusion section. The discussion also lacks information regarding the limitations and implications of the study. 4. There is no acknowledgment section in the manuscript. The author should acknowledge anyone who contributed to the study but did not meet the authorship criteria. 5. There are grammatical errors in the manuscript. The language needs to be improved.\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": "3933", "date": "30 Aug 2018", "name": "Liza Sari", "role": "Author Response", "response": "Response: This research has passed the ethics approval with number 501/H2.F1/Etik/2014. The Ethic committee is The Health Research Ethics Committee of the Faculty of Medicine, University of Indonesia. The chairman is Prof. Dr. dr. Rianto Setiabudy, Sp.FK. (The ethical approval is in the attachment). The HSC-3 and HSC-2 cell lines used in this study were provided by the Oral Biological Laboratory, Faculty of Dentistry of the University of Indonesia. We would like to thank Assoc Prof Masa-Aki Ikeda advisor in Japan who has provided them. We have sent all graphs of the flow cytometry, which are the original results of manual gating using CellQuest software (Becton Dickinson, NJ) to the F1000 Research editorial team. All the graphs contained in the article are the same graphs as the results of flow cytometry but have been saved in JPEG. Response: The study of apoptotic and caspase-3 activities of areca nut on the oral cancer cells, especially HSC-2 and HSC-3, was the novel or first research conducted as far as we know. The Previous study only has indeed tested ethanolic extract of areca nut cytotoxicity activity against the different type of cancer cells such as MCF-7 cells, and they didn’t count the number of cells undergoing apoptosis, so we don't have any information about other studies with the same form of this research. We also have studied the capability of areca nut in cytotoxicity activity on oral cancer cells and has been published in the other previous journal. We try to be very careful in comparing this study with other previous research especially if they were using different cell types and methods. Limitations of the study: The apoptosis activity using flow cytometry have several advantages, including fast period time analysis (thousand of cells per second), single cell analysis, and multiparametric measurements (correlations with several different cell events in one unit of time), but this machine also has drawbacks; the presence of physical and enzymatic manipulations during cell preparation and staining, can trigger additional apoptosis or necrosis cell numbers. Furthermore, flow cytometry is only used to calculate the number of apoptotic cells based on PS staining out of the cell membrane. That's why flow cytometry is more appropriate to detect early apoptosis. If the test aims to improve the accuracy of DNA fragmentation calculations in late apoptosis, it's recommended to use Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL). The previous study has indeed shown the existence of cell death through extrinsic pathways due to the inhibition of NF-kB and MAPKs by catechin derived from green tea. The discussion in this article is focused on caspase-3 activity as a determinant of the cell death. This study is part of a series of areca nut research that is still going on. The discussion of these proteins will be discussed in our next research. Acknowledgment: This study was a research without grants. This article was translated by Transmedical Institute. Proofreading process was done by Transmedical Institute. The writing technique has been corrected by Grammarly and during the revision process, the editorial team of F1000 research has also improved the sentence structure in the article." } ] }, { "id": "38222", "date": "25 Sep 2018", "name": "Masa-Aki Ikeda", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present article Liza Meutia Sari et al. examined the effect of areca nut extract on apoptosis in oral squamous cell carcinoma cell lines, HSC-2 and HSC-3 cells. They show that areca nut extract induced apoptosis in these cells by PI and Annexin-V staining followed by flow cytometry. They confirmed the results by detecting active caspase-3 activity following areca nut extract treatment. They conclude that areca nut extract induces apoptosis and caspase-3 activity in HSC-2 and HSC-3 cell. This is a limited but well-conducted study. However, there are several points that need to be addressed.\n\nSpecific points:\nChemosensitivity of cancer cells is determined by adding different concentrations of a drug. The authors should present the data regarding the effect of different concentrations of areca nut extract on cell viability, when they determined the IC50of HSC-2 and HSC-3 cells.\n\nAccording to Abstract and Methods, the authors carried out statistical analysis of their data. They should indicate which data are statistically significantly different in each graph.\n\nFigure 1B, 2B, 3B, and 4B: The chart legends should be \"Areca nut\" but not its concentrations. The concentrations should be written in Figure legends.\n\nFigure 1B, Top: 24 and 48 should be 24 h and 48 h, respectively.\n\nFigure 1B (right panel): It appears that many apoptotic cells (>50%) are detected and only less that 40% of cells are viable in control at 48 h, raising a question about the reliability of the results. The authors should clarify this point.\n\nFigure 2A and 2B (left panel): While more than 80% of cells are viable in control at 48 h, many apoptotic cells (>60%) are detected and only 25% of cells are viable in control at 24 h, raising a question about the reliability of the results. The authors should clarify this point.\n\nFigure 3B and 4B: By changing M1 and M2 to caspase-3 (-) and caspase-3 (+), respectively, readers will be able to understand the results more easily.\n\nDiscussion page 10, the middle of 1st para: The authors mentioned \"the percentage of HSC-2 cells undergoing apoptosis is high than HSC-3 cells. This result is possibly because there is a difference of cell sensitivity againt areca nut extract\". However, HSC-2 cells were treated with a higher concentration (629.5 ug/ml) of areca nut extract than HSC-3 cells (164.05 ug/ml). Because the IC50 of HSC-2 cells is higher than that of HSC-3 cells, HSC-3 cells appear to be more sensitive to areca nut extract than HSC-2 cells. This is confusing. The authors should clarify this point.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4069", "date": "26 Oct 2018", "name": "Liza Sari", "role": "Author Response", "response": "Chemosensitivity of cancer cells is determined by adding different concentrations of a drug. The authors should present the data regarding the effect of different concentrations of areca nut extract on cell viability, when they determined the IC50of HSC-2 and HSC-3 cells. Comment: Our preliminary study showed that the areca nut has a high content of total phenolic and flavonoid.1 The areca nut has chemosensitivity of cancer cells in different concentrations. We performed a MTS assay to observe the [G1] areca nut extract on cell viability. Five doses were adding into cancer cells, which were 160, 320, 640, 1280, and 2560 μg/mL in HSC-2, HSC-3, and HaCat cells.1 We found that the areca nut extract was cytotoxic towards HSC-2 (IC50 629.50 µg/mL), while in the HSC-3 cells, the IC50 is lower than HSC-2 cells (IC50 164.06 µg/mL). The areca nut showed weak cytotoxicity against HSC-2 cells. Sakagami et al. found that flavonoid-related phenols especially flavones showed weak cytotoxic activity against HSC-2. According to Abstract and Methods, the authors carried out statistical analysis of their data. They should indicate which data are statistically significantly different in each graph. Figure 1B, 2B, 3B, and 4B:The chart legends should be \"Areca nut\" but not its concentrations. The concentrations should be written in Figure legends. Figure 1B, Top:24 and 48 should be 24 h and 48 h, respectively. Comments: Thank you very much for the corrections, we have corrected all the figures as you instructed. Figure 1B (right panel):It appears that many apoptotic cells (>50%) are detected and only less that 40% of cells are viable in control at 48 h, raising a question about the reliability of the results. The authors should clarify this point. Comments: The flow cytometry analysis was performed to reveal the loss of plasma membrane asymmetry in cells. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein with high affinity for PS and binds to exposed apoptotic cell surface PS. Annexin V can be conjugated to fluorochromes while retaining its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells undergoing apoptosis[G1] . This is one of the earliest features of apoptosis. In our research, The flow cytometry was performed triple for both cells. The cells are processed with enzymatic degradation, centrifugation, and/or filtration to isolate the cells of interest, and the resulting cellular suspension is “stained” with fluorescent antibodies. When HSC-2 cells were cultured with areca nut for 48 hours, most of the cells were in the upper right quadrant; AV+/PI+. It means that most of the cells have undergone late apoptosis (Figure 1B). However, when HSC-2 cells were cultured for 48 hours under the same condition without areca nut treatment, we found that only less than 40% of the cells were viable. [G2] This condition suggests that the preparation of the staining process[G3] in flow cytometry itself may trigger the death of the cells (apoptosis or necrosis). This includes one of the limitations of our research.[G4] [G5] [G6] The same result is seen in the HSC-3 cells for 24 hours without treatment.Figure 3B and 4B:By changing M1 and M2 to caspase-3 (-) and caspase-3 (+), respectively, readers will be able to understand the results more easily. Comments: Thank you very much for the correction, we have made the corrections as you instructed Discussion page 10, the middle of 1st para:The authors mentioned \"the percentage of HSC-2 cells undergoing apoptosis is high than HSC-3 cells. This result is possibly because there is a difference of cell sensitivity againt areca nut extract\". However, HSC-2 cells were treated with a higher concentration (629.5 ug/ml) of areca nut extract than HSC-3 cells (164.05 ug/ml). Because the IC50 of HSC-2 cells is higher than that of HSC-3 cells, HSC-3 cells appear to be more sensitive to areca nut extract than HSC-2 cells. This is confusing. The authors should clarify this point. Comments: We have read a report from the previous article : 1. Kamiya Y, Ohshima T. The individual cell properties of oral squamous carcinoma and tumor suppressor gene mutation. Oral Sci Intl 2005;2(2):104-17.2. Sakai E, and Tsuchida N. Most human squamous cell carcinoma in the oral cavity contain mutated p53 tumor suppressor genes. Oncogen 1992;7:927-33. We think that it could be our limitation in exploring the characteristics of the HSC-3 cells, but maybe this explanation can open our mind about the result: This result is possible because of the characteristic of HSC-3 cells is different from HSC-2 cells. The HSC-3 cells have p53 gene mutation.3 The mutation of HSC-3 cells was confirmed in a previous report.4 However, when the p53 gene mutates, the mutated p53 protein is excessively produced or accumulated, thereby compromising apoptosis and leading to abnormal or malignant cell growth.5 We found that HSC-3 cells have the ability to withstand apoptosis higher than HSC-2 cells. However, this finding may vary by the study design and so much more data must be collected to better understand this phenomena." } ] } ]	1	https://f1000research.com/articles/7-723
https://f1000research.com/articles/8-989/v1	01 Jul 19	{ "type": "Case Report", "title": "Case Report: In-office bleaching, microabrasion, and resin infiltration for the correction of hypomineralized esthetic defects", "authors": [ "Ahmed Zakaria Aboelenein", "Mona Ismail Riad", "Mohammed Fouad Haridy", "Mona Ismail Riad", "Mohammed Fouad Haridy" ], "abstract": "Enamel hypomineralization is a condition that affects the quality of enamel, resulting in a change in its translucency and color. In this case report, a patient with a chief complaint of discolored front teeth, represented a very interesting case as he had combined opaque white, brown discoloration, and pitted enamel well distributed over the entire facial surfaces of enamel, especially his anterior teeth. It was also found that tooth #10 was protruded in relation to the adjacent teeth. The patient’s main concern was to improve his aesthetic appearance. Despite being a typical clinical picture of fluorotic lesion, fluoride was excluded as the cause of his lesions as the patient’s history indicated a lack of exposure to fluoride. A combined minimally invasive treatment, consisted of teeth bleaching, microabrasion, and resin infiltration were performed to address these esthetic problems. Minimum tooth reduction plus resin composite placement was done to solve the problem of the protruded tooth. All materials used was placed according to the manufacturer's instructions. Intraoral photographs were taken directly after each treatment to document any change in the appearance of the case. Six months follow up of the case was documented. The result was acceptable for the patient, and he was satisfied as more aggressive treatments were avoided.", "keywords": [ "Hypomineralization", "resin infiltration", "microabrasion", "bleaching" ], "content": "Introduction\n\nHypomineralization defects are considered one of the most common reasons patients seek esthetic dental treatment. However, management of such defects is always challenging as they imply proper identification of the cause, nature, severity of the defect, and proper understanding of all available treatment modalities that suites various degrees of the defect, thus achieving satisfactory results.\n\nThe probable etiological factors for enamel hypomineralization defects in permanent teeth can be broadly divided into two main categories, according to whether those defects have a localized or generalized distribution1. Localized hypomineralization defects could be caused by trauma, localized infection, and irradiation. While generalized (diffuse) hypomineralization may be caused by a wide range of factors1. Genetic disorders resulting from a single gene defect, as an X-linked, autosomal dominant or autosomal recessive trait are considered one of the factors2,3. Fluoride intoxication is one of the most common types of intoxications that causes enamel hypomineralization. Fluorotic lesions are characterized by opaque white spots or discolorations ranging from yellow to dark brown. The severity of those lesions depends on the duration and amount of fluoride intake during tooth development4. Perinatal and postnatal illnesses that may occur in premature and low birth weight neonates could be also responsible for the occurrence of enamel hypomineralization5. Infectious diseases and fever during early childhood such as chickenpox, measles, and mumps have also been linked to the occurrence of hypomineralization defects6.\n\n\nCase presentation\n\nA 27 year-old single Egyptian male, working as a clothes vendor, visited the outpatient clinic of Operative Dentistry Department, Faculty of Dentistry, Cairo University in March 2017 requesting to remove the discoloration from his front teeth. The patient was not satisfied with his smile because of the discolored teeth. He was healthy, with no systemic diseases. The patient’s dental history showed an irregular attendance to dental care with history of fillings, root canal fillings, and extraction.\n\nClinical examination showed a diffuse opaque white spots and streaks, together with subsurface brown discolorations and pitted enamel representing severe hypomineralization. Figure 1 illustrates the pre-operative photos of the case. This clinical appearance was very confusing concerning its cause, as it has long been considered a typical clinical picture of enamel fluorosis. Nevertheless, developmental defects of enamel with similar appearances are not necessarily caused by similar aetiologic factors.\n\n(a) Smile view. (b) Frontal retracted view. (c) Right side retracted view. (d) Left side retracted view. (e) Maxillary occlusal view. (f) Mandibular occlusal view.\n\nConversely, the same aetiologic factors can produce different defects at different stages of tooth development1. Consequently, not all white or brown hypomineralized enamel are caused by fluorosis7. In addition, the medical history of this patient was noncontributory regarding his exposure to fluoride, as this patient did not report that he had lived in an area where water is fluoridated during his childhood, nor had he taken any fluoride supplements, Thus, based on the patient’s history, fluoride was excluded to be the cause of hypomineralization.\n\nThe patient had calculus deposits on the upper left canine and premolar teeth, and bleeding on probing, thus full mouth debridement was performed together with instructions about maintenance of good oral hygiene. Besides, the clinical and diagnostic cast examination revealed a protrusion (malalignment) of tooth #10 relative to the adjacent teeth. In addition, teeth #21 and #32 were badly decayed. Tooth #21 was restored with a root canal treatment, ready- made post, resin composite core, and a definitive full coverage restoration. As for teeth # 32, root canal treatment, and a definitive composite restoration was made.\n\nPeriapical radiographs (Figure 2) showed that teeth #19 and #30 had periapical radiolucencies related to their defective root canal fillings. Tooth # 19 was extracted as it was unrestorable. While tooth # 30 was subjected to a re-treatment of its root canals, and a definitive composite restoration.\n\n(a) Periapical radiograph of tooth #19. (b) Periapical radiograph of tooth #30 (c) Periapical radiograph of tooth #30 after endodontic re-treatment and a definitive restoration.\n\nThe initial treatment plan for the hypomineralization defects was presented to the patient, this included in-office bleaching with 35% hydrogen peroxide (polaoffice in-office Tooth Whitening System, SDI Australia, 7700031) to target the brown discolorations, followed by resin infiltration to mask the white spot areas with Icon (DMG Germany, 220343). The patient was informed that the correction of the protruded tooth #10 would have to be delayed until after completion of the whitening and resin infiltration procedures.\n\nOne session of 3 applications of an in-office whitener (polaoffice in-office Tooth Whitening System, SDI Australia, 7700031) was performed for the patient, each of 8 minutes duration. The patient mentioned no post-treatment sensitivity, and no inflammation in his soft tissue was observed. The patient was informed that a period of 3 weeks was necessary between the bleaching sessions and the resin infiltration procedures. Figure 3 illustrates the clinical picture after the in-office bleaching, it was noticed that a residual light brown color still remains on the anterior teeth especially tooth #8. It was decided at this moment that a change in the initial treatment plan would have to occur, and that teeth microabrasion (Opalustre, Ultradent USA, 555) was going to be carried out before resin infiltration to selectively target those resistant stains.\n\n(a) Frontal retracted view. (b) Right side retracted view. (c) Left side retracted view.\n\nThe disappearance of those resistant stains was evident immediately after the microabrasion session (Figure 4). The patient returned to the clinic one week later for the resin infiltration procedures (Figure 5 & Figure 6). The patient was now told at this stage of the treatment plan that he would have the correction of his protruded #10 done two weeks after completion of the resin infiltration. All the materials used in this case were applied according to the manufacturer’s instructions. Table 1 illustrates the materials used in this clinical case, their composition, and catalogue number. Table 2 illustrates the timeline of the esthetic rehabilitation of the case.\n\n(a) Application of Opalustre microabrasion gel. (b) After microabrasion, frontal view. Notice the brightening of the brown stain that was present on tooth #8. (c) After microabrasion, right sided view. (d) After microabrasion, left sided view.\n\n(a) Icon-Etch gel (DMG Germany) was applied to the surface and left undisturbed for two minutes. The gel was thoroughly rinsed with water for 30 seconds. The teeth were then air dried with water- and oil-free air for 15 seconds. (b) Icon-dry (DMG Germany) was applied to the surface and left undisturbed for 30 seconds. The teeth were then air-dried with water- and oil-free air for 15 seconds. (c) Icon-Infiltrant (DMG Germany) was applied and left undisturbed for three minutes. Excess material was gently air-blown to prevent pooling around the incisal edge. (d) Excess resin was removed with dental floss. The resin was then light cured for 40 seconds in each tooth. Notice the difference between the resin infiltrated lateral, canine, and the other teeth.\n\n(a) Frontal view. (b) Right sided lateral view. (c) Left sided lateral view.\n\nAbbreviations: Bis-GMA, Bisphenol A diglycidylmethacrylate; TEGDMA, Triethyleneglycoldimethacrylate; BIS-EMA, Bisphenol A polyethylene glycol diether dimethacrylate\n\nA waxed up model (Figure 7) was fabricated and shown to the patient to express his opinion. The model included the following: a slight reduction (0.5 mm) of the mesio-incisal aspect of the labial surface of tooth #10, increase of the length of the clinical crown of tooth #10 with white wax, labial realignment of tooth #10 with resin composite, with a similar realignment for tooth #7 to preserve symmetry of the frontal aspect of the teeth.\n\n(a) Frontal view. (b) Palatal view.\n\nThe steps that was done on the model to realign tooth #10 was duplicated inside the patient’s mouth using a putty consistency guide (zetaplus, Zhermack, C 100600) fabricated from the waxed-up model. After the resin composite shade was selected and minimal tooth reduction (0.5 mm) of the mesio-incisal aspect was carried out as previously mentioned, enamel was then etched with 37.5% phosphoric acid gel (Kerr, Italia, 31297) for 15 seconds, rinsed with water for 10 seconds, and dried with air. OptiBond All In One adhesive (Kerr, Italia, 29670) was then applied for 15 seconds in a rubbing motion, and gently air dried followed by light curing (Elipar LED curing light, 3M ESPE) at an intensity of 1200 mw/cm2 for 10 seconds from the facial and palatal aspects. Resin composite was then applied in layers starting with A1 enamel shade (Herculite XRV Ultra, Kerr Italia, 34002) from the palatal aspect, followed by A2 dentin shade (Herculite XRV Ultra, Kerr Italia, 34019) to replace missing dentin and finally A1 enamel shade on the labial aspect. Each layer was light cured for 40 seconds from the both facial and palatal aspects. Finishing was carried out using a tapered flat end finishing carbide bur #7713 (MIDWEST, DENTSPLY 388529), and polishing was performed using fine, extra-fine discs (OptiDisc, Kerr 4182, 4183) and rubber cups (HiLuster Gloss PLUS Polisher, KerrHawe 2653 ). Figure 8 shows the procedure of realignment of tooth #10, and enhancing the final esthetic outcome using resin composite restorations. Figure 9 and Figure 10 show the final photographs of the case.\n\n(a) All prepared enamel was etched with 37.5% phosphoric acid gel for 15 seconds. (b) OptiBond All In One adhesive was applied for 15 seconds in a rubbing motion, air dried and then light cured. (c) Trimmed putty consistency index containing A1 enamel shade composite to be adapted on the palatal surface of tooth #10. (d) Tooth #10 having a palatal wall of A1 enamel. (e) Sectional matrix placement. (f) Tooth#10 after re-alignment, finishing and polishing.\n\nFinal clinical aspect of the case (a) Frontal retracted view. (b) Right sided retracted view. (c) Left sided retracted view. (d) Maxillary occlusal view. (e) Mandibular occlusal view. (f) Smile view.\n\n\nDiscussion\n\nThe aim of the esthetic rehabilitation in this case was to restore the patient esthetics and self-confidence in the most conservative way. The success of different treatment plans proposed for treating enamel hypomineralization cases depends on the severity of the defect. Most clinical reports aimed at conservative management of those defects have incorporated different interventions such as teeth bleaching, enamel macroabrasion, microabrasion, and resin infiltration in their treatment plans. The main difference between these reports is the sequence with which these interventions were used8–12.\n\nA recent clinical report8 has recommended masking enamel fluorosis stains using at home bleaching with 10% carbamide peroxide in an overnight tray as a first phase of treatment, followed by resin infiltration to mask the residual white spots. They did not recommend the use of in-office bleaching as it would cause post-treatment sensitivity. On the contrary, in-office bleaching was used in our case because the patient compliance to wear a tray was doubtful, additionally no sensitivity was reported after the treatment.\n\nThe second phase of the treatment in our clinical case included enamel microabrasion. This was opposite to a previous report8, who recommended resin infiltration as a second phase rather than microabrasion. They stated that resin infiltration would be more conservative as it removes only 40 µm of surface enamel, while microabrasion removes up to 200 µm of enamel corresponding to 10 applications. In our case, only 2 applications of microabrasion, which contains 6.6% HCl and silicon carbide, was carried out, which removed 50 µm of the enamel surface which is nearly equivalent to the amount removed during resin infiltration, and was sufficient in our case to remove those residual brown stains. In addition, the procedure of resin infiltration blocks the enamel from the labial surface because of the thin layer of resin coating the surface, and this would preclude the use of any further intervention from the labial surface (the most important surface). It is for this reason that the previous case report5 had to apply the bleaching gel from the lingual surface to target a residual yellow color after bleaching was carried out. Microabrasion also harmonized the color of the tooth after bleaching, and prepares the surface for resin infiltration.\n\nA previous case report9 has recommended macroabrasion as a first stage in treating hypomineralization defects. They used an ultrafine diamond bur (macroabrasion) followed by eight applications of the microabrasion gel. After one week, the microabrasion procedure was repeated, and finally three sessions of in-office whitening (35% hydrogen peroxide) were carried out. This approach was considered to be aggressive because of the enamel removal by macroabrasion.\n\nIn another case report10, two sessions of an in-office bleaching, three applications in each session was the starting protocol for such case. This was followed by 12 applications of the microabrasion material. This was consistent with the strategy adopted in our case, except that the number of applications of the bleaching material and that of the microabrasion material was quite big, so it may be considered also an aggressive treatment.\n\nRealignment of tooth #10 and #7 was carried out in our current case using minimum reduction (0.5 mm) of the mesio-incisal surface, increase in its clinical length, and re-shaping using resin composite. This was considered a perfect alternative for patients unwilling to undergo orthodontic treatment, or when ceramic restorations are not feasible. Resin composite was used to create an optical illusion that the protruded teeth were realigned, conforming to the shape of the arch, and the result was quite acceptable for our patient. Figure 10 illustrates six months postoperative evaluation of the patient and he was satisfied with the result.\n\n\nPatient perspective\n\nThe patient was pleased with the provided conservative treatments, he preferred the use of as few clinical procedures as possible. He was satisfied with the outcome and his self- esteem was improved.\n\n\nConclusion\n\nThe proper management of hypomineralization defects depends on the evaluation of the severity of the defect, and proper evaluation of the outcome after each single step of intervention rather than predetermined interventions based on anticipated outcomes. Thus, we consider in-office teeth bleaching, followed by microabrasion, and resin infiltration an acceptable method in treating hypomineralization discolorations.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details and 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": "Author contributions\n\n\n\nAhmed Zakaria Aboelenein: Data Curation, Investigation, Writing – Original Draft Preparation.\n\nMona Ismail Riad: Supervision, Writing – Review & Editing.\n\nMohammed Fouad Haridy: Project Administration, Writing – Review & Editing.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWong HM: Aetiological Factors for Developmental Defects of Enamel. Austin J Anat. 2014; 1(1): 1003. Reference Source\n\nWright JT, Hart PS, Aldred MJ, et al.: Relationship of phenotype and genotype in X-linked amelogenesis imperfecta. Connect Tissue Res. 2003; 44 Suppl 1: 72–8. PubMed Abstract \| Publisher Full Text\n\nGopinath VK, Al-Salihi KA, Yean CY, et al.: Amelogenesis imperfecta: enamel ultra structure and molecular studies. J Clin Pediatr Dent. 2004; 28(4): 319–22. PubMed Abstract \| Publisher Full Text\n\nRobinson C, Kirkham J: The effect of fluoride on the developing mineralized tissues. J Dent Res. 1990; 69 Spec No: 685–91; discussion 721. PubMed Abstract \| Publisher Full Text\n\nSeow WK: Effects of preterm birth on oral growth and development. Aust Dent J. 1997; 42(2): 85–91. PubMed Abstract \| Publisher Full Text\n\nMarshall JA: Dental Hypoplasia: Its Occurrence, Histopathology and Etiology. J Am Dent Assoc. Elsevier; 1936; 23(11): 2074–82. Publisher Full Text\n\nCutress TW, Suckling GW: Differential diagnosis of dental fluorosis. J Dent Res. 1990; 69 Spec No: 714–20; discussion 721. PubMed Abstract \| Publisher Full Text\n\nPerdiga J, Lam VQ, Burseth BG, et al.: Masking of Enamel Fluorosis Discolorations and Tooth Misalignment With a Combination of At-Home Whitening, Resin Infiltration, and Direct Composite Restorations. Oper Dent. 2017; 42(4): 347–56. PubMed Abstract \| Publisher Full Text\n\nPontes DG, Correa KM, Cohen-Carneiro F: Re-establishing esthetics of fluorosis-stained teeth using enamel microabrasion and dental bleaching techniques. Eur J Esthet Dent. 2012; 7(2): 130–7. PubMed Abstract\n\nBertassoni LE, Martin JM, Torno V, et al.: In-office dental bleaching and enamel microabrasion for fluorosis treatment. J Clin Pediatr Dent. 2008; 32(3): 185–7. PubMed Abstract \| Publisher Full Text\n\nBezerra-Júnior DM, Silva LM, Martins Lde M, et al.: Esthetic rehabilitation with tooth bleaching, enamel microabrasion, and direct adhesive restorations. Gen Dent. 2016; 64(2): 60–4. PubMed Abstract\n\nSammarco G: Combined minimally invasive treatment of white and brown fluorotic discolorations in a teenager: a case report. Int J Esthet Dent. 2019; 14(2): 148–55. PubMed Abstract" }	[ { "id": "50917", "date": "26 Jul 2019", "name": "Enrico Spinas", "expertise": [ "Reviewer Expertise Dental Traumatology", "Prosthetic Dentistry", "Paediatric Dentistry", "Orthodontics", "Conservative 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\nWe reviewed the article sent to us. In our evaluation, the organization is well-defined with a brief but complete introduction, interesting comparisons in the discussion and a precise presentation of the case. In this very last part however, we found a paragraph that we consider not appropriate at all in the context of the case discussion and is also somehow repetitive; thus, probably, it would sound better in the introduction. It would be necessary to insert some more recent citations. Lastly, a little grammatical mistake about the verb \"to be\" is present in the introduction.\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": "52646", "date": "23 Aug 2019", "name": "Michael J Wicht", "expertise": [ "Reviewer Expertise Cariology", "doctor-patient interaction", "decision-making process", "aesthetic rehbilitation", "bleaching", "resin-infiltration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors submitted a clinical case report about a full mouth rehabilitation of a young male patient exhibiting moderate to severe enamel defects and staining resulting in an unfavorable aesthetic appearance. The patient’s chief complaint was to enhance anterior teeth aesthetics and overall appearance. The hidden message was a negative self-image and restricted self-confidence mainly due to discoloration. The treatment plan consisted of a hygiene phase, in-office tooth whitening, micro-abrasion, resin infiltration and minor aesthetic corrections using composite restorations. On a different note, an endodontic re-treatment as well as the extraction of a so called un-restorable molar were performed.\n\nThe manuscript is well organized and the case is really worth to be reported because it shows, in an outstanding manner, that minimal interventions can result in excellent clinical results even from an aesthetic point of view and concomitantly enhance oral health and self-esteem.\n\nUnfortunately, the manuscript also contains numerous errors and suffers from shortcomings that are worth being addressed and revised accordingly. In the following, I’d like to itemize my suggestions for improvement:\nI would like to read more about the patient’s expectations. What exactly did he want to achieve, what would he be able to invest, were there any no-goes?\n\nAre there any anomalies regarding his anamnesis or family history? I have the feeling that there was something overlooked.\n\nDoes the patient smoke or regularly consume any discoloring foods or beverages?\n\nWhy is it important to mention that the patient is single? Please explain in detail or just leave it. To me it sounds slightly discriminating.\n\nFigure 2 A and B are inverted which is quite confusing.\n\nI would love to read more about the decision-making process. What alternatives were presented, why did the patient go for this option? Was he informed about risks and side-effects, if yes, which ones? Was the decision-making participatory or was it just a recommendation.\n\nTo me one, if not the, major success was the absence of gingivitis after treatment. Apparently, the full mouth debridement has been beneficial. However, and this should be taken into consideration as well, the patient was seemingly motivated to intensify his oral hygiene substantially. Was this effect stable over the observation period? The resolution was not high enough to comment on this from my side.\n\nIn the discussion part compliance should be replaced by willingness (second paragraph).\n\nThe literature falls short of the actual state of art. Please see Schoppmeier et al. 2018, da Cunha Coelho et al. 2019, di Giovanni et al. 2018 for more recent citations.1,2,3\n\nThe discussion is mostly oriented towards materials and methods used for operative care. This is fine; however, as I mentioned before the most precious aspect of this case to me is the cost-benefit analysis relative to Oral Health related Quality of Life. I would appreciate to see a focus also on that patient-centered outcome as well.\n\nI like the patient’s perspective part a bit more detailed.\n\nFinally, the manuscript needs linguistic revisions, it contains too many errors.\nIn summary, I encourage the authors to consider my thoughts in order to make this good case an excellent one depicting that modern dentistry can affect a great deal without sacrificing too much time, money and, first and foremost, tooth substance.\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-989
https://f1000research.com/articles/8-988/v1	01 Jul 19	{ "type": "Review", "title": "Tourette syndrome research highlights from 2018", "authors": [ "Olivia Rose", "Andreas Hartmann", "Yulia Worbe", "Jeremiah M. Scharf", "Kevin J. Black", "Olivia Rose", "Andreas Hartmann", "Yulia Worbe", "Jeremiah M. Scharf" ], "abstract": "This is the fifth yearly article in the Tourette Syndrome Research Highlights series, summarizing research from 2018 relevant to Tourette syndrome and other tic disorders. The authors briefly summarize reports they consider most important or interesting. The highlights from 2019 article is being drafted on the Authorea online authoring platform, and readers are encouraged to add references or give feedback on our selections using the comments feature on that page. After the calendar year ends, the article is submitted as the annual update for the Tics collection on F1000Research.", "keywords": [ "Tourette syndrome", "tic disorders", "review", "natural history", "etiology", "pathophysiology" ], "content": "Introduction\n\nThis article is meant to disseminate recent scientific progress on Gilles de la Tourette Syndrome (TS).\n\n\nMethods\n\nWe searched PubMed from time to time during 2018 using the search strategy “(”Tic Disorders“[MeSH] OR Tourette NOT Tourette[AU]) AND 2018[PDAT] NOT 1950:2017[PDAT]”. On 20 May 2019 this search returned 192 citations. Colleagues also recommended articles, and we attended selected medical conferences. We selected material for this review subjectively, guided by our judgment of possible future impact on the field.\n\n\nResults\n\nEpidemiology. All chronic tic disorders (CTD) begin at some point in time; it has not been clear which children with a recent-onset tic disorder would go on to develop a chronic tic disorder, and which would permanently remit. Kim and colleagues report on 43 children initially studied an average of 3 months after tic onset, of whom 39 returned at the 12-month anniversary of their first tic (Kim et al., 2018a; Kim et al., 2019). Not surprisingly, symptoms had generally improved at the 12-month mark. Surprisingly, however, every child still had tics, some apparent only when observed alone (by video). Baseline predictors of more severe tics at outcome included baseline tic severity, subsyndromal autism spectrum symptoms, three or more phonic tics, or an anxiety disorder. These results suggest that the current 12-month cutoff for chronicity may be arbitrary, and that mild tics that are not evident on a typical brief examination may be much more common than previously known.\n\nLowe and colleagues performed a very long interval follow-up study on TS, assessing tic severity, quality of life, and psychosocial function (Lowe et al., 2019). Of 150 surveys mailed to patients seen 25–32 years ago, 45 were returned (30%). 79% reported still having at least some tics, 40% reported some level of social impairment, and 20% were either unemployed, disabled or financially supported by family. However, the great majority of patients had improved in terms of tics and were doing well in other areas of their lives. Tics are 4-6 times more common in children with attention deficit hyperactivity disorder (ADHD) than in those without, and tics add to clinical problems and reduced quality of life (Poh et al., 2018).\n\nSensory phenomena and premonitory urges. Jakubovski et al. administered an online survey to characterize premonitory urges in individuals with TS (Jakubovski et al., 2018). The authors found that premonitory urges occurred in 73% of their sample, and that premonitory urge was more likely to occur with complex tics (78.6%) over simple tics (68.9%). Premonitory urges tended to localize in the same body area as the tics themselves, contrary to previous, smaller reports.\n\nInteroception is interesting as it shares features with sensory phenomena in TS (Khalsa et al., 2018). A derived measure of interoception predicts tic severity and premonitory sensations in TS; the authors propose their results suggest “a heightened higher-order sensitivity to bodily sensations in TS, relative to a noisier perceptual representation of afferent bodily signals” (Rae et al., 2019).\n\nImpaired olfactory function, but normal peripheral detection of olfactory stimuli, was demonstrated in TS (Kronenbuerger et al., 2018). Subjects with TS (n = 28) did not differ in odor detection threshold, but did present with impairments in both odor discrimination and odor identification. There was no significant difference in tobacco usage between the groups. 25% percent of subjects with TS met criteria for functional anosmia, compared to 7% of controls. This suggests that alterations in olfactory phenomena likely occur during higher-order processing, rather than during peripheral detection, in people with TS. This observation is consistent with previous similar conclusions about tactile sensory function in TS.\n\nOther. Weingarden et al. report that self-esteem is decreased in adults with TS and CTD (n = 122) (Weingarden et al., 2018). This, however, is less related to tics or tic severity but rather related to other psychiatric symptoms. When treating these patients, self-esteem was improved more by Comprehensive Behavioral Intervention for Tics (CBIT) than by psychoeducation and supportive therapy (PST), which seems plausible. It would be interesting to replicate the same study in children and adolescents. A review also examines the concept of self-esteem in TS and CTD, drawing a similar conclusion: that poor self-esteem appears more strongly related to psychiatric comorbidities than to tic severity, and, unsurprisingly, affects quality of life (Silvestri et al., 2018).\n\nTS phenotypes were investigated in 174 children and adolescents in French university clinic (Cravedi et al., 2018). Three clusters were identified. One of them corresponded to a tic-only phenotype (’pure’ TS) whereas another cluster included learning and intellectual disabilities, ASD, and ADHD. The third cluster corresponded to an ADHD profile with rather high intelligence and handwriting problems due to tics. Therapeutic implications are discussed.\n\nA survey of almost 700 parents, about half with tics, found that children with current or past tics slept an average of 1.5 hours less per night than control children (Ricketts et al., 2017). This result supports clinical experience and underlines both the impact of tic disorders on quality of life and a potential avenue for treatment. In children and adolescents with obsessive-compulsive disorder (OCD), religious symptoms were not associated with various clinical variables including outcome (Wu et al., 2018). This result suggests that scrupulosity and similar symptoms in children with TS can be expected to improve just as much as other OCD symptoms with appropriate treatment.\n\nWhile restricted food preferences have been well-documented in autism spectrum disorder (ASD), Smith et al. found similar patterns of food sensitivity in 30 children with TS. 25 subjects had comorbid conditions, though none in the sample had been diagnosed with ASD. Caregivers of children with TS reported their children having greater sensory sensitivities to food, greater food selectivity, and decreased consumption of dairy, vegetables, and fruit (Robbins, 2018; Smith et al., 2019).\n\nSevere neck tics led to a vertebral artery dissection (Aydin et al., 2018), a reminder that tics are not always benign. Another case report describes closed head trauma due to head banging in a 15-year-old, with important sequelae (Fasano & Galluccio, 2018). These reports underline the need for rapid and aggressive tic management in some patients.\n\nGenetics. There have been a number of advances in TS genetics over the past year, each arising from large-scale, multi-consortia collaborative efforts. Building off of two articles published the previous year reporting an increased genome-wide significant burden of rare, gene-disrupting mutations in 5–10% of TS patients (Huang et al., 2017; Willsey et al., 2017), Wang and colleagues reported the largest study of rare, de novo gene-disrupting mutations in TS to date (Wang et al., 2018). After first demonstrating that this class of mutations (de novo likely gene-damaging coding mutations and gene-disrupting copy-number variants/deletions) were specifically enriched in TS patients without a family history of a tic disorder (two unaffected parents) compared to patients with a positive parental history of tics, they conducted a combined de novo analysis of 802 parent-proband trios that identified a new high confidence TS susceptibility gene CELSR3, as well as an observation that the group potentially causative new mutations were more likely to affect genes involved in cell polarity. In parallel, Yu and colleagues reported the latest TS genome-wide association study (GWAS) of 4819 cases and 9488 controls, in which they identified one strong candidate TS gene (FLT3), and confirmed prior findings that a large proportion of TS heritability can be attributed to the aggregated effects of common genetic risk variants spread across the genome (Yu et al., 2019). They subsequently demonstrated that, in subjects with a family history of a tic disorder (TS or chronic tics), aggregated genome-wide TS polygenic risk scores (PRS) were significantly associated with lifetime worst-ever tic severity scores. In addition, both Yu and colleagues and Abdulkadir and colleagues used the TS GWAS polygenic risk scores to probe two independent population-based GWAS samples and found that individuals with non-TS tic disorders also have elevated TS polygenic risk compared to unaffected controls, albeit to a lesser degree than individuals with TS (Abdulkadir et al., 2019); (Yu et al., 2019). These two findings therefore confirm at the genetic level that TS and other tic disorders likely exist along a continuous spectrum as opposed to their current classification as distinct diagnostic entities. Lastly, Mufford and colleagues used the same TS GWAS PRS to probe imaging genetic data of subcortical brain volumes in the ENIGMA (Enhancing Neuro Imaging Genetics Through Meta Analysis) consortium, essentially tying the genetics of TS to the genetics of brain volumes (Mufford et al., 2018).\n\nSeparately, a huge study of shared GWAS data from over a million people with various neurologic and psychiatric disorders revealed that TS shares common variant genetic risk with OCD, major depression, and, unexpectedly, with migraine, especially migraine with aura (Brainstorm Consortium et al., 2018).\n\nEnvironmental risk factors. A large, full-population study from Sweden studied all singleton births in Sweden over a 30-year period, using siblings as controls (Brander et al., 2018). It found that “impaired fetal growth, preterm birth, breech presentation and cesarean section were associated with a higher risk of” TS or CTD. The risks were dose-dependent, with hazard ratios rising from 1.41 for one adverse perinatal event to 2.42 for five or more such events. This report is important due to its careful design, sample size and implications. It confirms previous indications that not all of the risk for TS is inherited, and points specifically to intrauterine and birth insults as contributing to that risk.\n\nA nationwide study from Denmark showed that children treated with an antibiotic or admitted to a hospital for an infection had a significantly higher risk of a later diagnosis of any psychiatric illness (Köhler-Forsberg et al., 2019). Interestingly, the highest risk for antibiotic use was for tic disorders, followed by OCD. (Hospitalization risk was higher for intellectual disability, though tic disorders were next highest.) The association does not prove that infections cause TS; e.g., patients destined to develop TS, or their parents, may be more likely to seek help for infections. Nevertheless, the association is interesting and deserves follow-up.\n\nAnimal models. Nespoli et al. (2018) found that dopaminergic imbalance in the dorsal striatum induced a Tourette’s-like phenotype in a rodent model. Administration of quinpirole, a selective D2/D3 receptor agonist, in juvenile rats with lesions to striatal projection neurons produced movements suggestive of both simple and complex tics in the neck, limbs, and mouth. A modified Yale Global Tic Severity Scale (YGTSS) was created to comprehensively score tic-like movements based on frequency, complexity, and severity of impairment. Immunohistochemical analyses revealed significantly decreased D1 receptor RNA expression at the lesion site, consistent with the decreased striatal D1 receptor expression seen in a human post-mortem study of TS (Lennington et al., 2016). The dopaminergic imbalance induced by decreased striatal D1 receptor activity, coupled with increased D2 receptor activity, may be relevant to TS.\n\nElectrophysiology. Eight awake TS patients undergoing DBS electrode implantation had recordings of individual cells of the external and internal globus pallidus (GP) (Israelashvili et al., 2017). Some cells in each division of the GP showed transient changes in firing rates associated with tics.\n\nNeuroimaging studies. Mahone et al. utilized 7T spectroscopy to investigate glutamate and GABA concentrations in children with TS. Findings included increased premotor area glutamate concentrations in TS, which also positively correlated with inhibitory control (Mahone et al., 2018).\n\nWhite matter tractography in children and adolescents with TS has not been well characterized, as previous studies utilizing diffusion tensor imaging (DTI) have primarily sampled from adult TS patients. Sigurdsson et al. examined white matter connectivity and water diffusivity in youth with TS, and report widespread decreases in axial diffusivity (Sigurdsson et al., 2018). The authors excluded subjects with high head motion, but even small head movements can affect DTI estimates (Baum et al., 2018).\n\nIn an event-related Functional magnetic resonance imaging (fMRI) study of 15 adults with TS, successful tic suppression correlated with increased activation in dorsal anterior cingulate cortex (van der Salm et al., 2018). 22 healthy controls performed a blink suppression task. During voluntary blink suppression, results yielded increased activation in ventrolateral prefrontal cortex, supplementary motor area, and cingulate motor area. These results suggest limbic system control of tic suppression, in contrast to sensorimotor system engagement in blink suppression. It would be an interesting follow-up to investigate whether suppression of normal blinks in TS patients displays the same patterns of activation seen in healthy controls, or reflects impairments in sensorimotor or limbic system control.\n\n21 adults with TS and 21 healthy controls performed an fMRI study during perception of neutral or angry faces (Rae et al., 2018). In TS, insula functional connectivity (fc) was increased with pre-supplementary motor area (SMA), premotor cortex, primary motor cortex and putamen. Insula fc with globus pallidus and thalamus varied with tic severity, while insula-SMA fc varied with premonitory sensations. These results strengthen the evidence that insula and SMA are involved with premonitory urges and tics.\n\nPharmacological studies. Maia & Conceição (2018) elaborate on their theory of how dopamine may relate to tics in TS. Hienert et al. (2018) provide a meta-analysis of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) studies in TS measuring the dopamine transporter (DAT) or D2-like dopamine receptors; neither showed conclusive differences in patients, but sample sizes and methodological limitations may explain the negative results.\n\nClinical and neuropsychological studies. The potential role that autoimmunity may play in TS pathology has long been controversial. A large-scale population study from a Swedish birth cohort assessed the risk factors of 40 different autoimmune diseases in individuals with TS/CTD and/or OCD, as well as family members with varying degrees of relatedness (Mataix-Cols et al., 2018). TS/CTD was associated with a 36% increase in likelihood of having any autoimmune disease, compared with matched population controls. Additionally, the mothers of subjects diagnosed with TS/CTD were 40% more likely to have an autoimmune disease; full siblings of individuals with TS/CTD carried a 17% increase in risk. The authors note that it is undetermined if these numbers support the hypothesized immunological component of TS/CTD, or instead reflect a selection bias for those already receiving medical care.\n\nA research consortium in Germany has recently proposed that tics may correspond to altered perception-action binding (Beste & Münchau, 2018). Petruo et al. (2018) now provide a first experimental demonstration in 35 adolescents with TS and 39 healthy controls using a Go/No-go task and subsequent electroencephalography (EEG) analysis, providing support for the idea that stimulus-action binding is stronger in patients with TS, and that “unbinding” may thus represent a useful therapeutic venue.\n\nInhibitory control in TS has been assessed in a myriad of different ways, with conflicting results. For instance, Mancini et al. (2018) report abnormal inhibitory function in patients with OCD, but no deficits in the TS group. Similarly, Stenner et al. (2018) performed a clever study of a form of automatic motor inhibition. Exposure to a subliminal stimulus at an appropriate time prior to a supraliminal movement cue inhibits movement, a phenomenon called the negative compatibility effect (NCE). Here, 21 adults with TS and 21 tic-free controls performed similarly, suggesting that NCE is another form of experimental motor inhibition that appears normal in TS.\n\nBy contrast, the role of abnormal habit-learning has been posited as a cognitive marker of TS. It is yet unclear if tics would result from increased habit-learning, deficits in unlearning maladaptive behaviors, or a combination of both. Shephard et al. (2018) investigated implicit sequence learning in youth with TS with or without ADHD compared to tic-free youth with or without ADHD. Subjects with ADHD demonstrated impaired learning of a motor sequence learning task, while subjects with TS had mildly enhanced learning. However, they found evidence of “hyper-learning,” in which subjects with TS had difficulty in unlearning the motor sequence task. Similarly, Kim et al. (2018b) demonstrated slower unlearning (forgetting) of a visuomotor task in children and adolescents with TS. The role that impaired habit unlearning may play in TS warrants further investigation.\n\nAdaptive functioning, a person’s ability to function and organize themselves in daily life, has not been well-characterized in TS. Taylor et al. found that, in a TS sample, deficits in adaptive functioning were mostly driven by impaired executive function (Taylor et al., 2018). ADHD and two or more comorbid conditions were associated with decreased adaptive functioning. More than half of the variance in this study was explained by deficits in executive function, rather than tics themselves. These results suggest that aggressive treatment of ADHD and comorbid diagnoses may be important in clinical management of TS.\n\nPsychological interventions. Group-based psychotherapeutic interventions for tics bear the promise of reduced costs and easier access to appropriate care, and several recent reports address this treatment option. One recent paper investigated the long term effects of group therapy on tic severity, quality of life and school attendance in 28 children with TS 12 months after completing habit reversal training (HRT) training or education (a follow up to the 2016 study (Yates et al., 2016)). This study demonstrated positive effects in the long run but apparently without significant differences between both groups (Dabrowski et al., 2018). A Danish study investigated a combined exposure and response prevention (ERP)/HRT protocol comparing group with individual sessions (n = 27 per group, n = 54 total). The efficacy on decreasing tic severity was similar in both treatment arms (Nissen et al., 2019). Traditionally, ERP sessions (as compared to HRT/cognitive behavioral intervention for tics (CBIT)) lasted for two hours, making them more difficult and expensive. In this study, session duration was shortened to one hour and shorter exposure was as effective, if not more, than the classic format (van de Griendt et al., 2018). In another potential method for wider behavior therapy accessibility, (Andrén et al., 2019) report preliminary results from a major study on therapist-supported, parent-guided, online behavior therapy for TS. Patients improved substantially (mean 75% with ERP, 55% with HRT), and the improvement persisted up to 12 months later. The average therapist time was only 25 minutes per week.\n\nMedication. Swedish treatment registries were searched to identify patterns of medication prescribing for almost 7000 patients with TS/CTD from 2005–2013 (Carulla-Roig et al., 2018). Among other interesting findings, ADHD drugs, antidepressants, and hypnotics/sedatives were all prescribed more often than antipsychotics, for which there is much stronger evidence of efficacy. Rizzo et al. (2018) provide the first direct comparison of pharmacotherapy with behavioral therapy in children and adolescents with TS / CTD. Both approaches were effective in reducing tics and improving quality of life; however, only pharmacotherapy was effective in reducing OC symptoms.\n\nThe D1 receptor antagonist ecopipam was compared to placebo in a double-blind, crossover, randomized controlled trial (RCT) in children and adolescents with TS (Gilbert et al., 2018). YGTSS total tic score (TTS) declined significantly more with the active drug. Another drug acting largely on dopamine, the VMAT2 inhibitor valbenazine, was tested in a phase IIb study in TS, but failed to meet the primary efficacy endpoint (Neurocrine Biosciences Inc., 2018).\n\nNeurosurgery. A large-scale public database and registry for deep brain stimulation (DBS) Tourette syndrome has been established (Martinez-Ramirez et al., 2018). This report summarizes information on 185 Tourette patients from 10 countries. Mean improvement in total YGTSS score was 40% at 6 months after vs. before surgery, and 45% at 12 months. The difference between stimulation sites (centromedian nucleus (CM-Pf), anterior globus pallidus interna (GPi), posterior GPi) was not statistically significant. About a third of patients had side effects, mostly related to stimulation not surgery. A large, collaborative group is using this database to investigate the optimal site for DBS via a 3D analysis (Johnson et al., 2018). DBS in children remains controversial. Coulombe et al. (2018) review available data on DBS in “children” (ages 12-21), and Smeets et al. (2018) discuss ethical considerations regarding DBS in TS patients under the age of 18.\n\nOther treatment. In a pilot study, Behler et al. administered bilateral cathodal transcranial direct current stimulation (tDCS) over motor cortex in three patients with TS. While one patient displayed a 35% improvement in tic severity, the other two subjects experienced worsening of symptoms. However, all three patients did report a decrease in negative affect, as well as a mild increase in positive affect (Behler et al., 2018).\n\nHimle et al. (2018) review how tics can affect the family and vice versa. Two large studies have now demonstrated clearly how much TS can hinder school performance. The first is a large study, conducted by the CDC via telephone survey, from the US. The authors found that TS (specifically, when compared to children without TS both with and without other neuropsychiatric conditions) has negative consequences for school performance (Claussen et al., 2018). Subjects with moderate-to-severe TS (n = 97), when compared to those with milder symptoms (n = 203), were significantly more likely to require an individualized education plan (IEP). Furthermore, the prevalence of co-morbid psychiatric illness in TS is quite high; 80% of subjects with TS had at least one co-morbid neuropsychiatric disorder, compared to 18% of subjects without TS. The second study is from the population registry in Sweden, demonstrating academic underachievement in TS/CTD across all educational levels (Pérez-Vigil et al., 2018). One limitation of this study is that it relies on those who are diagnosed by a physician, who we know are a proper subset of those with tics. One would not be surprised to learn that the factors that lead to care-seeking (like severity of symptoms, or other undiagnosed behavioral problems) may also themselves interfere with education. Commenting on this article, Hartmann and Delorme write, “Ticcing as such impairs attention, and attempts to supress tics at school or work makes things even worse. Teasing and bullying because of tics is a further contributing factor to attention problems. Thus, tics impair academic achievement in their own right, and comorbidities can only partially be blamed for this situation. … TS and CTD are not harmless neurological conditions but profoundly affect a person’s life trajectory (see also Mataix-Cols’ work on suicide in TS (Fernández de la Cruz et al., 2017)). Take tics seriously!” (Hartmann & Delorme, 2018).\n\nSeveral useful review articles appeared during 2018 (Efron & Dale, 2018; Ganos et al., 2018; Garnaat et al., 2018; Hartmann & Worbe, 2018; Stern, 2018).\n\n\nConclusions\n\nThe literature on TS is increasing rapidly. Fortunately, studies with larger sample sizes are becoming more common. Several simple but important questions remain to be answered in TS, including the following: Why do tics tend to start at ages 5–10? Why are they more common in boys? Why do they tend to improve during sleep? Why do tics usually improve in early adulthood? How accurately can we predict outcome for an individual patient? Which patients need which treatments? Is secondary prevention possible? Hopefully future studies will address these and other important issues.", "appendix": "Grant information\n\nThis work was supported in part by the U.S. National Institutes of Health (NIH) [R01 MH104030].\n\nThe authors confirm that the funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAbdulkadir M, Mathews CA, Scharf JM, et al.: Polygenic Risk Scores Derived From a Tourette Syndrome Genome-wide Association Study Predict Presence of Tics in the Avon Longitudinal Study of Parents and Children Cohort. Biol Psychiatry. 2019; 85(4): 298–304. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrén P, Aspvall K, Fernándezde la Cruz L, et al.: Therapist-guided and parent-guided internet-delivered behaviour therapy for paediatric Tourette’s disorder: a pilot randomised controlled trial with long-term follow-up. 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PubMed Abstract \| Publisher Full Text\n\nSigurdsson HP, Pépés SE, Jackson GM, et al.: Alterations in the microstructure of white matter in children and adolescents with Tourette syndrome measured using tract-based spatial statistics and probabilistic tractography. Cortex. 2018; 104: 75–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilvestri PR, Baglioni V, Cardona F, et al.: Self-concept and self-esteem in patients with chronic tic disorders: A systematic literature review. Eur J Paediatr Neurol. 2018; 22(5): 749–756. PubMed Abstract \| Publisher Full Text\n\nSmeets AYJM, Duits AA, Horstkötter D, et al.: Ethics of Deep Brain Stimulation in Adolescent Patients with Refractory Tourette Syndrome: a Systematic Review and Two Case Discussions. Neuroethics. 2018; 11(2): 143–155. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith B, Rogers SL, Blissett J, et al.: The role of sensory sensitivity in predicting food selectivity and food preferences in children with Tourette syndrome. Appetite. 2019; 135: 131–136. PubMed Abstract \| Publisher Full Text\n\nStenner MP, Baumgaertel C, Heinze HJ, et al.: Intact automatic motor inhibition in patients with tourette syndrome. Mov Disord. 2018; 33(11): 1800–1804. PubMed Abstract \| Publisher Full Text\n\nStern JS: Tourette’s syndrome and its borderland. Pract Neurol. 2018; 18(4): 262–270. PubMed Abstract \| Publisher Full Text\n\nTaylor C, Mitchell-Blake T, Kharod A, et al.: Predictors of adaptive functioning in children with tourette syndrome attending a specialist clinic. Arch Dis Child. 2018. Reference Source\n\nWang S, Mandell JD, Kumar Y, et al.: De Novo Sequence and Copy Number Variants Are Strongly Associated with Tourette Disorder and Implicate Cell Polarity in Pathogenesis. Cell Rep. 2018; 24(13): 3441–3454.e12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeingarden H, Scahill L, Hoeppner S, et al.: Self-esteem in adults with Tourette syndrome and chronic tic disorders: The roles of tic severity, treatment, and comorbidity. Compr Psychiatry. 2018; 84: 95–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWillsey AJ, Fernandez TV, Yu D, et al.: De Novo Coding Variants Are Strongly Associated with Tourette Disorder. Neuron. 2017; 94(3): 486–499.e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu MS, Rozenman M, Peris TS, et al.: Comparing OCD-Affected Youth With and Without Religious Symptoms: Clinical Profiles and Treatment Response. Compr Psychiatry. 2018; 86: 47–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYates R, Edwards K, King J, et al.: Habit reversal training and educational group treatments for children with Tourette syndrome: A preliminary randomised controlled trial. Behav Res Ther. 2016; 80: 43–50. PubMed Abstract \| Publisher Full Text\n\nYu D, Sul JH, Tsetsos F, et al.: Interrogating the Genetic Determinants of Tourette’s Syndrome and Other Tic Disorders Through Genome-Wide Association Studies. Am J Psychiatry. 2019; 176(3): 217–227. PubMed Abstract \| Publisher Full Text\n\nvan de Griendt JMTM, van Dijk MK, Verdellen CWJ, et al.: The effect of shorter exposure versus prolonged exposure on treatment outcome in Tourette syndrome and chronic tic disorders - an open trial. Int J Psychiatry Clin Pract. 2018; 22(4): 262–267. PubMed Abstract \| Publisher Full Text\n\nvan der Salm SMA, van der Meer JN, Cath DC, et al.: Distinctive tics suppression network in Gilles de la Tourette syndrome distinguished from suppression of natural urges using multimodal imaging. Neuroimage Clin. 2018; 20: 783–792. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "50717", "date": "15 Jul 2019", "name": "Lorena Fernández de la Cruz", "expertise": [ "Reviewer Expertise Obsessive-compulsive disorder", "Tourette's syndrome", "body dysmorphic disorder", "hoarding disorder", "psychiatric epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is yet again a good summary of the most relevant TS literature published in the past year (2018). The text is useful for researchers and clinicians in the TS and related fields, and in my opinion constitutes a nice educational piece. The included papers are overall well chosen and summarised. I only have a few minor comments, listed below.\nThe authors have included in their review the paper by Wu et al. (2018)1, which refers to the clinical characteristics and treatment outcomes of children and adolescents with OCD with and without religious symptoms. This article concludes that the presence of religious symptoms is not associated with OCD treatment outcomes. Based on this, the authors of the current review affirm that this suggests that scrupulosity and similar symptoms in children with TS can be expected to improve just as much as other OCD symptoms with appropriate treatment. While this may be true, I think this conclusion is long stretched based on the Wu et al. (2018) data, which actually does not refer to TS or tics at all. I therefore suggest to remove this paper from the review.\nWhen the authors mention the treatment study by Andrén et al. (2019)2, I think it is important to mention that the percentages in the parenthesis in the sentence “Patients improved substantially (mean 75% with ERP, 55% with HRT)” actually refer to treatment responders, and not the percentage of patients that simply improved.\nLast sentence of the “Etiology” section: “the association is interesting and deserves follow-up”. This reads weird to me and I suggest to change the wording to “deserves to be further explored” or similar.\nPlease check the manuscript for typos; there are quite a few. Some of them are listed below:\n“Phenomenology and natural history” section; “Sensory phenomena and premonitory urges”, third paragraph: the verb is missing in the sentence “alterations in olfactory phenomena ARE likely to occur…”\n\n“Phenomenology and natural history” section; “Other”, second paragraph: “in A French university clinic” instead of “in French university clinic.”\n\n“Pathophysiology” section; “Neuroimaging studies”, third paragraph: write “functional” in lower case.\n\n“Treatment” section; “Neurosurgery”: a registry for deep brain stimulation (DBS) FOR Tourette syndrome…”\n\nNote that the acronym ASD appears before it is spelled out, two paragraphs later (page 3).\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? Partly\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": "52244", "date": "02 Sep 2019", "name": "Marc Lavoie", "expertise": [ "Reviewer Expertise Neuropsychology", "Cognitive electrophysiology", "Clinical psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well selected review that covers most of the essential 2018 works on Tourette syndrome. One area that will probably have a big impact in the future, is the ability to predict which neurocognitive variables represents good predictors of clinical evolution in TS. Electrophysiological measures were useful to predict outcome, following a psychological intervention, in other psychiatric disorders, such as anxiety disorders, depression and obsessive-compulsory disorder (Krause et al., 2015; Burkhouse et al., 2016)1,2, but has yet to be tested in TS. One article did just that last year. I suggest to add an article by Morand-Beaulieu and colleagues (2018)3 that suggest that electrophysiological markers could offers new predictors for therapeutic outcome in TS and CTD patients with its high temporal precision to follow the stream of fast cognitive and motor processes. Morand-Beaulieu et al (2018) suggested that motor-related and slow cortical potentials could constitute electrophysiological markers of TS. These measures are related to SMA and basal ganglia functioning which is pretty consistent with neuroimaging and electrophysiological studies already reported in the current manuscript. I believe that this article could fit well in the ‘’clinical and neuropsychological studies’’ section or perhaps in the ‘psychological intervention’ section if you decide to integrate it.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-988
https://f1000research.com/articles/7-1742/v1	02 Nov 18	{ "type": "Case Report", "title": "Case Report: Exertional rhabdomyolysis in a spin class participant with sickle cell trait", "authors": [ "Teresa Longo", "Matthew Shaines", "Teresa Longo" ], "abstract": "Exertional rhabdomyolysis is more common in sickle trait due to a predisposition to dehydration and inability to concentrate the urine. Spinning, an indoor cycling workout, has been associated with exertional rhabdomyolysis in recent reports. A consequence of rhabdomyolysis is acute kidney injury, which may be expected to be more common in patients with sickle trait. We report a case of spinning induced rhabdomyolysis in a woman with sickle trait that did not result in renal injury. “Spin rhabdo” is thought to be more severe than other causes of exertional rhabdomyolysis and is associated with higher creatine kinase levels than other causes of exertional rhabdomyolysis. Therefore, individuals with known sickle trait should visit their physician prior to participation in spin classes for the first time. We might also consider voluntary screening for sickle trait in at risk populations prior to enrolling in spin classes given that many patients are unaware of their sickle trait status.", "keywords": [ "Rhabdomyolysis", "Spin class", "Sickle cell trait" ], "content": "Introduction\n\nRhabdomyolysis is a clinical syndrome consisting of intense muscle breakdown and necrosis. It is caused by a variety of triggers, including trauma, infection, drugs, and intense exertion. Nontraumatic exertional rhabdomyolysis is more common in patients with disorders of glucose and lipid metabolism or sickle cell disease, although it may occur in individuals with normal muscles if exertion is significant enough to impair muscle oxygenation. Spinning is a high intensity stationary cycling exercise performed in a group setting and often synchronized to music. It has recently been linked to case reports of exertional rhabdomyolysis in otherwise young, healthy individuals1.\n\nOne of the major complications of rhabdomyolysis is acute kidney injury (AKI), caused by a combination of volume depletion and accumulation of myoglobin pigment released by damaged muscle cells inside the renal tubules. Measurement of creatine kinase (CK) levels in the serum is an important indicator of muscle necrosis2; a recent study found that CK levels are higher in spinning associated rhabdomyolysis than in other causes of exertional rhabdomyolysis3. This finding implies the degree of muscle necrosis may be higher in spinning induced rhabdomyolysis, a correlation that is especially concerning for individuals predisposed to renal injury. Sickle trait, while usually considered benign, may predispose patients to renal problems such as hematuria and hyposthenuria. Here, we report a case of “spin class rhabdomyolysis” treated with aggressive intravenous hydration without acute kidney injury in a young woman with sickle cell trait.\n\n\nCase report\n\nA 28-year-old Hispanic female with no significant medical history presented to the emergency department with acute onset bilateral leg pain and dark urine 3 days after attending a spin class for the first time. She did not regularly participate in high intensity workouts, although she did frequently walk for exercise. The class consisted of 45 minutes of high intensity cycling. She did not drink much water before or during the class. Several hours after the class, her legs felt weak and gave out. She endorsed bilateral thigh pain that she described as heavy, sore, and limiting her activity. One day prior to admission she noticed that her urine was brown in color, which prompted her to present to the emergency department.\n\nShe never experienced similar symptoms in the past. She denied sick contacts, recent travel, recent illness, fevers or feeling overheated, significant alcohol or drug use, or history of deep vein thrombosis. She took no medications and had no allergies. Although she had one episode of pyelonephritis several years prior to presentation, she reported no personal or family history of renal disorders. Family history was unremarkable for known genetic disorders, musculoskeletal disorders, or metabolic myopathies.\n\nVital signs were stable upon presentation. Her thighs were tender to palpation and strength of lower extremities was limited by pain. Dorsalis pedis pulses were palpable but weak. There was large blood on urine dipstick and minimal red blood cells on urine microscopic analysis. Creatine kinase (CK) was 74,978 IU/L and she was admitted with a diagnosis of rhabdomyolysis. Renal function was normal. She was treated with aggressive intravenous isotonic saline and sodium bicarbonate. Upon discharge, CK was decreasing and she was able to tolerate large intake of oral fluids. A hemoglobin electrophoresis pattern was performed at outpatient follow up to search for a potential underlying cause of her rhabdomyolysis. The result was consistent with sickle cell trait: HbA 57.8% and HbS 37.6%.\n\n\nDiscussion\n\nMany factors can predispose to rhabdomyolysis, including trauma, metabolic disorders, sickle cell disease, dehydration, high temperature, medications, and excessive exercise4. There has been an increase in the number of admitted patients with exertional rhabdomyolysis in recent years5. While sickle cell trait is usually considered benign, a 2016 study found a significant association between sickle trait and exertional rhabdomyolysis in U.S Army soldiers6. Sickle trait may predispose to dehydration due to an inability to concentrate urine and conserve water in conditions of strenuous exercise, which leads to lactic acid buildup and erythrocyte sickling7.\n\nSpinning is a high intensity, indoor bicycle sport often synchronized to music that has been growing in popularity. It has been associated with exertional rhabdomyolysis most commonly in young, otherwise healthy females. The majority of cases occur in women under the age of 35 years8. While cases of “spin rhabdo” occur overwhelmingly in first time spin class participants with deconditioning, patients often have no other predisposing factors. Rhabdomyolysis may occur after as little as 15 minutes of spinning9. Spectrum of illness is variable and ranges from mild to severe with potential for life threatening complications such as AKI and compartment syndrome1. One study suggested that patients with spinning induced rhabdomyolysis showed more severe disease and longer length of hospital admission than patients with rhabdomyolysis of other causes8. Additionally, a 2016 retrospective cohort study found that spinning induced rhabdomyolysis was associated with higher CK levels than other causes of exertional rhabdomyolysis3.\n\nThe risk of AKI is thought to be lower when CK values are less than 15-20,000 IU/L on admission10. Multiple studies have shown a weak correlation between serum CK value and incidence of AKI, and renal injury has occurred at CK levels as low as 5,000 IU/L10. This correlation has one major implication, namely that individuals with sickle cell trait should be especially cautious when participating in spin classes for the first time. The higher CK levels associated with spinning induced rhabdomyolysis may be particularly concerning for individuals with risk factors for kidney disease, such as those with sickle trait. Therefore, we recommend that individuals with known sickle trait visit a physician prior to participation in spin classes and discuss ways to minimize their risk of rhabdomyolysis.\n\nIn many parts of the world, newborn screening to detect both sickle cell disease and trait is widely available. In the United States, all states are now mandated to offer screening; while most parents opt to have their children tested, reporting of the result to families is variable. In a 2006 survey, only 16% of respondents were aware of their sickle trait status11. A number of sports organizations advocate for universal screening prior to participation in high intensity athletics due to rare reported instances of exercise related sudden death12. In 2010, the National Collegiate Athletic Association (NCAA) implemented a mandatory opt out sickle trait screening policy prior to athletic participation13. However, screening in adults remains controversial due to the potential for loss of employment, insurance, and other forms of discrimination based on sickle trait status. This risk must be weighed against the potential benefits from screening, which include the opportunity for providers to discuss preventative measures with patients. Given the severity of spinning-induced rhabdomyolysis and its increasing incidence in recent years, we may consider voluntary screening for sickle trait in at risk populations prior to enrolling in spin classes for the first time. Our patient was unaware that she had sickle trait; rhabdomyolysis may have been prevented had she been screened and educated about the importance of hydration during high intensity exercise.\n\n\nConclusion\n\nGiven the longer length of hospital admission and higher CK values associated with spinning induced rhabdomyolysis, individuals with known sickle trait should talk to their primary care physician before participating in spin classes. Prevention of exertional rhabdomyolysis may require more aggressive hydration to maintain fluid and electrolyte balance in individuals with sickle trait than in normal spin class participants. Organizers of spin classes should take precautions to ensure novel participants hydrate well and do not overexert themselves during the class. Voluntary screening for sickle trait in at risk populations prior to enrolling in spin classes may be considered given many individuals are unaware of their sickle trait status.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nRamme AJ, Vira S, Alaia MJ, et al.: Exertional rhabdomyolysis after spinning: case series and review of the literature. J Sports Med Phys Fitness. 2016; 56(6): 789–93. PubMed Abstract\n\nBaird MF, Graham SM, Baker JS, et al.: Creatine-kinase- and exercise-related muscle damage implications for muscle performance and recovery. J Nutr Metab. 2012; 2012: 960363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCutler TS, DeFilippis EM, Unterbrink ME, et al.: Increasing Incidence and Unique Clinical Characteristics of Spinning-Induced Rhabdomyolysis. Clin J Sport Med. 2016; 26(5): 429–31. PubMed Abstract \| Publisher Full Text\n\nBosch X, Poch E, Grau JM: Rhabdomyolysis and acute kidney injury. N Engl J Med. 2009; 361(1): 62–72. PubMed Abstract \| Publisher Full Text\n\nAalborg C, Rød-Larsen C, Leiro I, et al.: An increase in the number of admitted patients with exercise-induced rhabdomyolysis. Tidsskr Nor Legeforen. 2016; 136(18): 1532–6. PubMed Abstract \| Publisher Full Text\n\nNelson DA, Deuster PA, Carter R 3rd, et al.: Sickle Cell Trait, Rhabdomyolysis, and Mortality among U.S. Army Soldiers. N Engl J Med. 2016; 375(5): 435–442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDincer HE, Raza T: Compartment syndrome and fatal rhabdomyolysis in sickle cell trait. WMJ. 2005; 104(6): 67–71. PubMed Abstract\n\nKim YH, Ham YR, Na KR, et al.: Spinning: an arising cause of rhabdomyolysis in young females. Intern Med J. 2016; 46(9): 1062–8. PubMed Abstract \| Publisher Full Text\n\nBrogan M, Ledesma R, Coffino A, et al.: Freebie Rhabdomyolysis: A Public Health Concern. Spin Class-Induced Rhabdomyolysis. Am J Med. 2017; 130(4): 484–487. PubMed Abstract \| Publisher Full Text\n\nKenny JE: Creatine Kinase: How Much is Too Much? The NYU Langone Online Journal of Medicine. 2010. Reference Source\n\nTreadwell MJ, McClough L, Vichinsky E: Using qualitative and quantitative strategies to evaluate knowledge and perceptions about sickle cell disease and sickle cell trait. J Natl Med Assoc. 2006; 98(5): 704–710. PubMed Abstract \| Free Full Text\n\nBlinder MA, Russel S: Exertional sickling: questions and controversy. Hematol Rep. 2014; 6(4): 5502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcDonald MA, Creary MS, Powell J, et al.: Perspectives and Practices of Athletic Trainers and Team Physicians Implementing the 2010 NCAA Sickle Cell Trait Screening Policy. J Genet Couns. 2017; 26(6): 1292–1300. PubMed Abstract \| Publisher Full Text" }	[ { "id": "44105", "date": "25 Feb 2019", "name": "Gregory Kato", "expertise": [ "Reviewer Expertise Sickle cell 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 a fairly balanced presentation of a case report of a case of exertion-related rhabdomyolysis and its potential association with sickle cell trait (SCT). There is suggestive evidence for association of exertion-related rhabdomyolysis with SCT, but only with assumed prevalence of SCT in military personnel of self-identified African descent. There has never been a well-controlled analysis of this sickle cell risk, but it is strongly suspicious based on extensive circumstantial evidence. There also is an extensive history in the 1960's of stigma and discrimination involving SCT that is not well-appreciated in the current generation of biomedical personnel.\nThe authors do a good job of presenting the issues involved in this case report with the exception of three points:\nAt least half of the cases of exertional rhabdomyolysis do not involve SCT, and so SCT is not a complete explanation of risk. Universal precautions help to address this population outside of SCT.\n\nSCT is highly prevalent and the vast majority of SCT subjects never develop rhabdomyolysis, including in spin classes, and additional risk factors must contribute to these rare cases.\n\nThere are inadequate data to base evidence-based recommendations and more research is needed in this area.\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": "45794", "date": "02 Apr 2019", "name": "Nyamkhishig Sambuughin", "expertise": [ "Reviewer Expertise Inherited muscle disorders", "genetic cause of rhabdomyolysis", "exertional rhabdomyolysis in SCT" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIndividuals with Sickle Cell Trait (SCT), generally a benign carrier state of hemoglobin mutation, are thought to be at increased risk for exertional rhabdomyolysis. This report presents an association between SCT and rhabdomyolysis triggered by spinning. The association between SCT and exertional rhabdmyosis is mostly based on case reports. There are no well-designed case-control study that address this association except epidemiological study in US army soldiers. In many case reports, including this one, authors often highlight SCT is a main contributing risk factor. However, etiology of rhabdomyolysis is extremely variable. Although this case report describes an event of rhabdomyolysis in sufficient details, other potential contributing factors were not well discussed. The subject in the report appears to be unaccustomed to intense exercise and was not adequately hydrated before and during exercise. These are well known factors that contribute to rhabdomyolysis. Combination of those factors will certainly increase subjects' risk to muscle necrosis. Individuals should be aware of these factors before enrolling in unaccustomed strenuous exercise, regardless if they have SCT or not.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-1742
https://f1000research.com/articles/7-1156/v1	30 Jul 18	{ "type": "Research Article", "title": "Emerging Hand Foot Mouth Disease in Bangladeshi Children- First Report of Rapid Appraisal on Pocket Outbreak: Clinico-epidemiological Perspective Implicating Public Health Emergency", "authors": [ "Md. Azraf Hossain Khan", "Kazi Selim Anwar", "A. K. M. Muraduzzaman", "Md. Abid Hossain Mollah", "S. M. Akhter-ul-Alam", "Kazi Munisul Islam", "Sheikh Ariful Hoque", "Md. Nazrul Islam", "Md. Ahasan Ali", "Md. Azraf Hossain Khan", "A. K. M. Muraduzzaman", "Md. Abid Hossain Mollah", "S. M. Akhter-ul-Alam", "Kazi Munisul Islam", "Sheikh Ariful Hoque", "Md. Nazrul Islam", "Md. Ahasan Ali" ], "abstract": "Background: Hand, foot and mouth disease (HFMD) is a common contagious disease among children under 5 years, particularly in the Asia-Pacific-region. We report a localized outbreak of childhood HFMD for the first time from Bangladesh, diagnosed only based on clinical features due to gross lack of in laboratory-diagnostic facilities. Methods: Following the World Health Organization’s case-definition, we conducted a rapid-appraisal of HFMD among 143 children attending Pabna Medical College and General Hospital with fever, mouth ulcers and rash. Data were collected between September and November 2017 using a preset syndromic approach and stringent differential diagnostic-protocols. Results: The mean age of children was 2.9±2.3 years. Age did not differ with sex (P=0.98), first sibling being more likely to (62%) belong to middle-income families. Younger children (<5 years) were more likely to suffer with moderate-to-high (38.5°C) fever (P<0.04), painful oral ulcers (P<0.03) and painful/itchy rash (P<0.01). Sex did not differ with other symptoms, but boys had less painful oral ulcers than girls (P<0.04). Fever (63%) and chicken-pox-like-rash (62%) was observed more in mid-October to mid-November than September to mid-October (P<0.01 and P<0.03, respectively). No differences in symptoms (fever, oral ulcers and extremity rash) were observed with precipitation, nor with ambient temperature. Children <5 years (85%) had quicker recovery (within 5 days) than those ≥5 years (69%), (P<0.04), with marginal differences in sex (P<0.05). Conclusions: Our findings highlight the potential usefulness in diagnosing HFMD based on clinical parameters, although stringent differential diagnosis remains indispensable. It is particularly applicable for resource-constrained countries who lack appropriate virology laboratory equipment. Since no specific treatment or effective vaccination is available for this disease, supportive therapy and preventive measures remain the primary methods to circumvent transmission augmented by climate-related factors. Standardized virology laboratory warrants appropriate diagnosis and globally representative multivalent vaccine is deemed essential towards preventing HFMD.", "keywords": [ "Emerging Childhood-HFMD", "Bangladesh", "Rapid-Appraisal", "Pocket-Outbreak" ], "content": "Introduction\n\nOf all commonly occurring febrile illness and rash syndromes1, hand, foot and mouth disease (HFMD) remains the most among young children2,3. Although this viral infection remains largely contagious4,5, it is self-limiting and benign. Severe cases occur red with a low incidence (3.2% to 8.5%) and fatalities are rare6–8. Starting in the West during the mid-1970’s1,2 HFMD emerged in the Asia-Pacific region in mid-1990s9–11 heralding as a major public health hazards2,10. Epidemiologically, it follows a 2–3 years cyclical pattern11 but may break out anytime9 as has occurred in India (Orissa12 and Calcutta13), bordering with Bangladesh.\n\nWith the complaints of mild-to-moderate fever (≥38.5°C8; 101.3°F) childhood HFMD, characteristically manifest with body rashes1,4, mostly of the knees and buttocks4,14, augmented by painful oral/buccal ulcers and blisters. Papulo-vesicular rash in the extremities consequently forms pustules6. Most children recover/heal within 7–10 days5,8,9. Of the few complications, neuro-respiratory syndromes4 (encephalitis, aseptic meningitis and acute flaccid paralysis)3,4 occur mainly in younger children; these are rare but seldom fatal9,15. Children are mostly affected by neurotropic viruses like enterovirus (EV71)2,16 and coxsackievirus (CA-6, A-10, A-16)5,7,10. These viruses are transmitted15,17 through direct contact/blister-fluid, droplets, oro-fecally16 and also spread out through contaminated environment, water and food18.\n\nReportedly, clinical diagnosis of HFMD is usually established depending on physicians’ suspicions13,14 as the sole diagnostic modality12. The diagnosis is primarily based on history of illness, disease-onset, presenting clinical-features1,6,19 and, socio-demographic profile12,14,20. Small erythematous maculopapular lesion (1–5 mm) enlarge (3–15 mm) and progress to vesicular eruptions with a prominent erythematous halo13,21. It is essential to perform stringent differential diagnosis (DD) to distinguish HFMD from a group of diseases. DD includes chickenpox, scabies, measles, erythema multiforme, herpangina, herpetic gingivitis, drug eruption and others4,14,17,22. Laboratory diagnosis is usually not essential12,19,23, and has been described by the World Health Organization (WHO) as optional1. Conversely, the sophisticated laboratory tests used for definitive diagnosis (virus isolation, molecular analysis, PCR, genotyping)1,13,24 are not available in most resource-constrained countries3,12,13 like Bangladesh.\n\nSince there is no specific treatment4,19,22,25 for HFMD, care largely remains palliative19 with antipyretics/analgesics and antihistamines. Topical anesthetics are rarely used for oral ulcers for soothing and comfort. Povidone-iodine used as a mouth wash/topical application that can relief pain. Since no effective vaccine against HFMD-viruses is available1,2,7, preventive measures remain the primary method of circumventing HFMD transmission to break infection-chains (droplets, oral-fecal route, and direct contact)2,18. Effective prevention requires personal hygiene, hand washing26 and a pollution-free environment12 including food and water18,27. Meteorological variations in precipitation8,9 and ambient temperature20,28 often impact on HFMD occurrences5 in the Asia-Pacific region5,9,10,15,17, along with atmospheric pressure and the relatively higher humidity in summer and early autumn18.\n\nExtracts from extensive reviews, when compared with our intensive observations on upsurge of unusual febrile, rash-associated childhood illnesses between July and August 2017, were indicative of HFMD. A rapid appraisal was therefore, designed as a short-term standardized-surveillance1. Following a pre-set case-definition and syndromic approach (according to the WHO HFMD guidelines1), similar to a study conducted in Thailand29 a strategic plan was adopted to conduct this comprehensive study from September to November 2017.\n\n\nMethods\n\nUtilizing a pre-set syndromic approach based on case-definition following the WHO’s HFMD guidelines1 this rapid appraisal (descriptive study) was conducted among 143 children attending Pabna Medical College and General Hospital (PMC-GH) from its catchment areas between September and November, 2017. PMC-GH is a 250-bed secondary care hospital serving a targeted population of nearly 2.81 million from its 2,371.5 km230 catchment area situated in a small poverty-stricken north-western flood-prone plain land on the Ganges Delta basin in Bangladesh.\n\nClinical diagnostic tool. Prepared based on syndromic case-definition following the WHO’s HFMD guidelines1, similar to a prior study conducted in Thailand29. Most of the contents of this tool have been shown in Figure 4 (4 A), showing the algorithm of Clinical diagnosis of HFMD1.\n\nClinical case management protocol. This was prepared incorporating a history of disease, onset, chief complaints and duration of illness, clinical diagnosis and therapeutic intervention. We ascertained clinical outcome by through post-treatment follow-up in the outpatient department of PMC-GH or through cellphone-based enquiry. We performed the clinical diagnosis following WHO guidelines1, predominantly based on three main signs/symptoms: fever, oral ulcers and rash in extremities. Fever was graded into moderate-to high (38.5°C) and none-to-low (37-38.4°C), oral ulcers were grouped into three stages- more painful, less painful and painless; and, rashes in extremities into three types: painful and itchy, painless and itchy; and painful but not itchy.\n\nSince pain remains subjective in younger children in expressing pain intensity properly, we arbitrarily categorized the pain intensity based on following clinical grounds:\n\ni. Nullifying any history of past disease/disorders that may confound the current pain status\n\nii. Facial expression of a child having body rash and/or oral ulcer on touch/other sensitizations\n\niii. Impression and/or opinion of child’s parent/guardian in respective cases\n\niv. Finally, clinician’s judgements based on disease history and presented signs/symptoms\n\nA therapeutic index was prepared to treat childhood HFMD cases following standard therapeutic plan consisting of: antipyretic/analgesics, antihistamines, anesthetic-cream for topical applications. These aforementioned three clinical diagnostic tools (moderate-to-high fever, painful oral ulcers and painful rash in extremities) have been prepared adopting the WHO clinical management and public health response for HFMD1 with a little modifications to suit our short-term comprehensive study (Figure 4 (4 A)).\n\nThis tool consisted of socio-demographic variables and household (HH) income. We categorized the income of children’s family following World Bank Data Help Desk 201631 as follows:\n\n- Low-income group: HH/families earning a monthly income of ≤6,946 BDT\n\n- Lower-mid income group: HH/families earning a monthly income of 6,947–27,336 BDT\n\n- Upper-mid-income group: HH/families earning a monthly income of 27,337–84,564 BDT\n\n- High-income group: HH/families earning a monthly income of ≥84,564 BD\n\nWe calculated income scale using the USD rate: 1US $ = 84.31 BDT as of 11 June 2018.\n\nRecords on seasonal data on local weather/climate (average temperature and rain precipitation) were collected from Pabna Meteorology Dept., Bangladesh over the period of September through November 2017. In Bangladesh, early autumn runs from September to mid-October, followed by late autumn/fall from mid-October to mid-November. All tools were pre-tested for this rapid-appraisal (small-scale disease surveillance)1,2.\n\nCrosschecked data were subjected to Pearson’s chi-squared test, Fisher’s exact test and Spearman correlation analysis using SPSS for Windows v.21, taking P<0.05 as indicating statistical significance (at 95% CI).\n\nAny child, irrespective of age and sex, attending PMC-GH between September and November 2017 with suspected HFMD (meeting WHOs1 recommended criteria) were included in this study. Suspected cases having other serious disease/co-morbidities were excluded, although patients were referred to concerned department for proper clinical management.\n\nFollowing standard procedure of ethical issues32, written informed consent was obtained from the parents of children with suspected HFMD prior to enrolment. We detailed the parents/guardian of all children on the purpose and procedures of this study. We also informed the parents on the lack of risk of harm/damage involved in procedures and did not collect body fluids or other biological samples. We informed the parents that they could remove their child at any stage of the study. Complete privacy and anonymity of clinical data was ensured, including its protected use research purposes only. This study had prior approval through the Ethical Committee of Pabna Medical College and General Hospital, Government of the Peoples’ Republic of Bangladesh (Memo No. 1577, dated: 26/08/2017).\n\n\nResults\n\nThe mean (±SD) age of the 143 children was 2.9±2.3 years; 80 (56%) were boys and 63 (44%) were girls. Of the total, 70% were under 5 years old. Age did not differ with sex (P=0.98). Data on HH structure yielded an average size of children’s family as 5.5±6.9 persons/per HH. Of them, 62% having only one (no siblings) and 38% two (first sibling) children, (Table 1).\n\nFollowing World Bank Data Help Desk, 201633\n\nFollowing Word Bank, (2016) standard31 family/HH income-group evidenced that majority families (85%) belonged to middle-income HH/families (34% belonged to upper-middle and 51% to lower-middle income-groups living with a modest HH budget). The rest (14.7%) belonged to low-income groups lived with a tight HH-budget. Notably, children from mid-income-HHs contracted significantly more HFMD which was more among the first siblings (P<0.01), (Table 1).\n\nChild’s age was significantly associated with three major clinical signs/symptoms. Younger children (under 5 years old) suffered more (74/91, 81%) with moderate-to-high fever than older children (17/91, 19%; p<.04). Similarly, painful oral ulcers (82/111, 74%) and painful itchy rash in extremities (92/116, 79%) were more common in younger than older children (p<0.03 and p<0.01, respectively). Notably, characteristics of skin rash in extremities of younger children’s were more predominantly papulo-vesicular (59/68, 87%) than chicken-pox-like (43/75, 57%), (P<0.01). However, sex did not differ with other signs/symptoms except oral ulcers: boys had less painful ulcers (23/32, 72%) than girls (9/32, 28%), (P<0.04), (Table 2).\n\nMean ± SD = 2.9±2.3.\n\nNone of the three major signs/symptoms of HFMD (fever, oral-ulcers/blisters and extremity rash) was associated with seasonal variations except fever and characteristics of rash. Moderate-to high fever (57/91, 63%) was observed more in fall/late-autumn (mid-October through mid-November) than in early autumn (September through mid-October), yielding 37% of cases (34/91), (p<0.01). Similarly, papulo-vascular rashes were more common in fall (42/68, 62%) than in early autumn (26/68, 38%) (P<0.03; Table 3).\n\na Comparatively lower temperature: Arbitrarily set cut-off values of lower temperature (on average). b Comparatively higher temperature: Arbitrarily set cut-off values of higher temperature (on average)\n\nThe three major sign/symptoms among these HFMD contracted children were more prevalent on days where 0.0 mm precipitation was recorded. Rain had no significant impact on any of the three major sign/symptoms, unlike on dry days with no rainfall (0.0 mm). Similarly, all major sign/symptoms prevailed more in hot and humid days when the ambient temperature was recorded at ≥30°C (up to a maximum of 36.2°C), with no significant difference among three major sign/symptoms (Table 3).\n\nFindings of post-treatment clinical outcome was associated with age. More younger children (<5 years) recovered in <5 days (63/74, 85%) than older peers (≥5 years) (47/69, 69%) who were more likely to recover in >5 days) (P<0.05). However, clinical disease/outcome was not associated with children’s sex, although boys were more likely to suffer with the illness for 6–7 days, whereas girls tended to recover within 5 days. However, this was only marginally significant (P<0.05; Table 4).\n\n\nDiscussion\n\nWe conducted an extensive review on HFMD in the latest literature. Clinico-epidemiological insight from these articles augmented by our careful observations and intensive appraisal on WHO’s “Clinical management and public health response for HFMD”1 enabled us to establish a primary clinical diagnosis of childhood HFMD on an unusual events of febrile-rash cases (M.A.H.K., unpublished observations, June–July 2017). Concurrent agreement from similar reports attested our HFMD diagnosis among those febrile illness and rash syndrome cases in children as correct1–3. Gauging the potential of a sudden upsurge in childhood HFMD cases (during July 2017) attending PMC-GH from its catchment area made us aware that we faced an upcoming localized outbreak. A strategic plan was thus urgently adopted to conduct this rapid appraisal (short-term standardized surveillance)1 on childhood HFMD utilizing a pre-set case-definition/syndromic approach based on the WHO’s HFMD guidelines1. This study is comparable with a study conducted in Thailand29.\n\nThe main objective of this descriptive study (rapid appraisal) was to create awareness on childhood HFMD as a newly emerged disease in Bangladesh and tended to stir-up country’s public health emergency squad in getting alert to combat similar upcoming-HFMD outbreaks. In conducting this study, we were also able to assess the clinical skill, diagnostic potential and epidemiological insight of PMC-GH team along with instant local support, which will also prove helpful. The findings of our study will help to show how skillfully the clinicians at the mid-level secondary-care hospitals remain capable in combating localized HFMD outbreaks quite confidently. They accomplished it utilizing own resources and work force, without seeking additional assistance. Credit goes to concerned physicians (dermatologist and/or pediatricians) in establishing correct diagnosis of childhood-HFMD based on strong yet rational suspicions, as reported by others12,13,23 along with institution of supportive therapy. Every issue was addressed and resolved successfully despite huge constraints in manpower, funding and gross lack in diagnostic facilities, though laboratory-test is reportedly not essential, often14,19,25.\n\nHFMD has emerged as a major public health problem in recent years2,10. HFMD was first recognized in the Western world1,2 during the mid-1970s2. It was then spread out in the Asia-Pacific region since the mid-1990s3,6,9, mostly in four countries (Malaysia, Taiwan, China and Singapore)3,6,9,11. HFMD outbreaks were also reported in the Indian districts of Orissa12 and Calcutta13, bordering with Bangladesh. It is therefore strange that no data or published reports exist in Bangladesh yet then, as Prabir et al. commented rightly and in-time23. Despite epidemiological forecasts that HFMD outbreaks occur in a 2–3-year cyclical pattern11, two large epidemics broke out in 2 consecutively years: one in Malaysia during 1997 and the other in Taiwan, the following year9.\n\nAll these facts and figures, including epidemiological hunches and variabilities support our strong speculation of this localized outbreak of HFMD in Pabna that we could combat boldly. Based on such experience, we do suspect that HFMD might have emerged in Bangladesh earlier, but, swept on unnoticed. HFMD often remains ‘underestimated’ due to its benign nature and self-limiting clinical features6,8. These facts led us to suspect that latent HFMD cases or small localized outbreaks might remained under-reported or un-reported (Kazi Selim Anwar and Md. Abid Hossain Mollah, personal observation, June 2017).\n\nUsing observations of clinical course, disease progression, short-term suffering and disease outcome confirms that childhood-HFMD remains a benign and self-limiting disease, observations that are consistent with several other studies6–8. We attest that HFMD can be diagnosed accurately once there is a strong suspicion13,14. The presenting signs and symptoms can be the sole diagnostic modality, too12. We diagnosed these HFMD cases based on a patient’s history, onset and the presented clinical features6,19. In addition, we considered the patient’s socio-demographic characteristics12,14, and a positive history of similar sign/symptoms in child’s family, nursery and/or in schools12,14. However, our data does not agree to such higher incidences of sever disease (8.5%) that was reported from Vietnam8, rather it goes in favor of ‘no’ or ‘rare fatalities’ contrararily5,6.\n\nOur data yielded a significant association between age groups and three major clinical signs/symptoms. Moderate-to-high fever, painful oral ulcers and itchy-painful rashes were directly proportional to younger children which was consistent with several other findings4–6,8,9. Moderate-to-high fever remains an important, but not mandatory or principal sign of HFMD, as the WHO’s guidelines for clinical and public health response indicate1, in agreement with our findings. Oral and/or axillary temperature in 64% of cases revealed a moderate fever (38.5°C), ranging mostly between 37.5°C and 38.2°C; the rest (36%) had no or a low-grade fever (ranging between 37.0 to 38.4°C). This variation in body temperature led us to postulate that fever itself can never be considered as the sole symptom in confirming HFMD diagnosis. Our observation on ‘fever’ though remain consistent with several authors, such as Van Pham et al.8, and other studies reported high patient body temperatures in HFMD-cases5,9.\n\nThe most important characteristic symptoms for HFMD remains papulo-vesicular rash, often manifesting as painful chicken-pox-like rashes. We observed that this papulo-vesicular rash (Figure 3) was painful in 60% of cases and was less painful/painless in 40% of cases. Since pain remains subjective in younger children in expressing pain intensity, we categorized HFMD based on no recent history of pain status, facial expression of child having body rash/oral ulcer on touch/other sensitizations and impression of child’s mother and the clinician’s rational judgements. Our findings on rashes and its types in patients with childhood HFMD remain consistent with other reports1,4, particularly its distributions in knees or buttocks1,2,7,13,14,17. We observed itchy rashes in child’s extremities, forming small pustules that were filled with turbid fluid (Figure 1) and in some cases it consequently crusted off6 after 3–4 days. These rash symptoms remain consistent with several prior reports1,4,13,14.\n\nMost of the children under 5 years (78%) suffering from HFMD had characteristic oral ulcers and/or painful blisters in tongue/mouth (Figure 2), symptoms cited in several reports1,4,7,14,16,17. However, we found less painful/painless oral ulcers/blisters in 22% HFMD cases. Although the exact reason for these less pain/painless oral ulcers remain unclear, we postulate that it could be due to a varied perception and/or different tolerability level by those children. Of course, being unwilling to mention or disclose about their tolerable/bearable little pain feeling shy or even being scared of cannot be ruled-out, either. Some of these children may have taken analgesics at home before coming to hospital, which they did not disclose despite repeated questioning. Notably, we did not find any sign/symptom that significantly differed with sex of children except oral ulcers. More boys had less painful ulcers than girls (P<0.04). Our findings that revealed a sex differences for some specific clinical sign/symptom remain unique, though findings from a study in India reported an overall male-female ratio of 21:1713.\n\nWe performed a thorough DD to make the clinical diagnosis of HFMD as perfect as practicable. We performed the DD to differentiate HFMD from closely similar diseases, like varicella/chicken-pox, scabies, measles, erythema multiforme, herpangina, herpetic gingivitis, drug eruptions, as several reports have mentioned1,6–8,22. Mosquito bite was also included in the DD as it was reported in a recent study in India, underlining this simple yet valuable DD-point13. Particular attention in the DD was paid to the characteristics of skin lesions where macules and papules quickly evolve into vesicles. Characteristically, the lesions in these children occurred on their palms, soles, and buttocks22. We observed vesicles in majority of these children that ruptured with the formation of erosions and crusts. However, Sharma et al. observed in Indian patients that it starts with small (1–5 mm) erythematous maculopapular lesions that rapidly enlarged to 3–15 mm lesions, progressing to vesicular eruption with prominent erythematous halo13,14. We observed this common dermatological phenomenon in some of our studied children with HFMD. Nonetheless, our observation remains similar to that of Bhumesh et al. from India that oral lesions begins as erythematous macules and then evolved into vesicles (measuring 2–3 mm) on an erythematous base21.\n\nLaboratory diagnosis is usually not essential12,23 to confirm a readily diagnosed HFMD case based on rational judgement of existing clinical features. Even, lab diagnosis often remains unnecessary19. Laboratory tests, such as serotyping, molecular, PCR and genotyping3 and virus culture1,13,24, may not be feasible, available and more importantly not affordable in resource-constrained countries12,13 like Bangladesh, particularly in hard-to-reach/remote areas. Although few studies report high WBC count or blood glucose, as associated with HFMD severity13–19,23,24, it remains scarcely seen in recent literature. Furthermore, rise in blood glucose level may be due to other viral diseases rather than HFMD, and may well remain confounded by a wide range of infections and/or inflammatory processes. Moreover, in some pediatric cases, it may not be practically possible to collect intravenous blood from younger children, who possess thin veins, particularly at the primary care health centers in grass-root level. Children often become too agitated when attempts are made to draw blood, as we observed, with their parents distressed, and the children non-compliant and non-cooperative. However, we observed this was more an issue among less-educated and low-income group families.\n\nLatest literature shows that virological diagnosis remains the main diagnostic tool. Of the four species in the family of Picornaviridae (groups EV-A, B, C and D) that cause HFMD in children, chiefly remain EV 71 and coxsackie-virus A-6, A-10 and A163,7,8,10,24. More crucial is that these viruses are transmitted rapidly15,17 through direct contact, respiratory droplets, via feces/blister-fluid and contaminated environment18.\n\nThere is no specific treatment22,25 or pharmacological intervention4 available for HFMD yet. Since it largely remain supportive19,25 we prepared a standardized therapeutic index involving our clinical experts that we followed as therapeutic measure among childhood cases of HFMD in this study. It consisted of: i) antipyretic/analgesics, ii) antihistamines, and iii) anesthetics drugs (oral gel or ointment). Skin lesions in those cases (observed in only two cases) healed within 3–4 days; we did not prescribe any acyclovir due to the highly reported adverse effects (nephropathy and neurotoxicity). Since oral acyclovir is poorly absorbed, we prescribed it as an exception in recommended dosage of oral syrup (20 mg/kg body-weight) for 5 days only in eight severe cases (mean age, 2.4 years). However, children with profuse skin-lesions with severe pain responded dramatically, with early recoveries. Reasons for this mechanism or the basis of its pathogenicity and the pharmaco-dynamics of acyclovir are not fully understood, which necessitates further investigation.\n\nThough no effective vaccine available yet against HFMD-viruses1,2,7. Scientists have been attempting to develop a vaccine against HFMD in Malaysia (since 2010)33, in China (since 2012)35, and in Taiwan (since 2014)36,37. Cai et al. demonstrated how active immunization with an experimental inactivated CA16 vaccine can confer full protection- which provided a solid foundation for developing inactivated whole-virus vaccines against CA16 infection in humans35. Similarly, Chih-Wei Lin et al.36 found some ‘prospect and challenges’ with critical bottlenecks in the development of multivalent HFMD vaccines. They demonstrated how combined vaccine would reduce number of injections simplifying WHOs ongoing child immunization schedule to protect against several viruses, like H5N1, EV71 and JEV at the same time36. Yican Cui et al. attempted to develop a combined bivalent-vaccine comprising EV71 and A16 to give balanced protective immunity37. There is also evidence for the further development of multivalent vaccines for broader protection against HFMD37.\n\nDue to a lack of available vaccines against viruses that cause HFMD15,17,18, preventive measures remain the primary way of circumventing HFMD. Prevention methods includes good personal hygiene, proper hand washing26 pollution free environment12,18, sewage27, and germ-free water and food18. Although avoiding person-to-person contact2 through isolation remain justified, it may often not be practical in unprivileged low-income communities and/or resource-constrained healthcare settings like Bangladesh. It is imperative to increase mass awareness among such communities more.\n\nIn agreement with other studies4,6, our data also revealed that younger children (<5 years old) recovered more quickly (in <5 days) than their elder peers (>5 years old) who recovered in 6-7 days (>5 days) (P<0.05). There was a marginal significant difference in sexes, since boys had seemingly quicker recovery than peer girls did (P<0.05). Nevertheless, according to latest literature, most childhood HFMD-cases resolve themselves within 7–10 days5,8,9. These findings remain consistent with that of other reports from Asia-pacific countries4–8, including India12,13,14,18.\n\nComplications of childhood HFMD remain few, although younger children may develop them more often7,17,21. We found three cases (2.09 %) of such complications (mild-to-moderate severity) who we had to pay a special attention to. The first case (a 4-year-old girl) was a case of pneumonia, who we treated with IV antibiotics and discharged following recovery after 2 days. The second one was an admitted case of pyoderma (a 5-year-old boy), who received appropriate antibiotics and was discharged on after 3 days. We diagnosed a third case of a 1.5-year-old girl with a case of post-HFMD Onychomadesis38, who had clinically diagnosed HFMD 25 days before, who had shedding of skin on her right little finger since last few days. On repeated observations (weekly) her nail resumed in original position after 3 weeks of her development of a nail problem without any medication. This scenario remains comparative to a report from South Korea38. However, the mechanisms of Onychomadesis and its association with HFMD is not yet fully understood as literature shows38 and that, some viruses are responsible for onychomadesis as a temporal variation.\n\nAlthough CA16 and EV712 are mostly associated with neuro-respiratory syndromes1,4, we did not observe serious complications, nor encountered any death in the children with HFMD, a finding that remain consistent with several reports5,6,9,15.\n\nSeveral studies carried out in the Asia-Pacific region reported an association of HFMD cases with a wide range of meteorological findings (weather, climate, ambient temperature, humidity, rain, etc.)5–10,15,17,18. Meteorological factors reportedly remain associated with HFMD outbreaks in Asia-Pacific regions5,17, like Singapore9,15, China10 and Hong Kong20. The rainy season8 and short-term variations in temperature20,28 had an impact on HFMD occurrence5 in this region. This includes atmospheric pressure, relative humidity and rain precipitation9 as well which peaks in summer and early autumn18. We conducted this study during autumn, that runs in Bangladesh from early September through November in two phases: early autumn from September to mid-October, while its next phase (late autumn/fall) from mid-October to November.\n\nOne limitation is that we could not conduct a proper meteorological study as reported from some Asian countries9,10,15,20. As a small part of our study, we only tried to find out briefly if local weather has any impact on HFMD just to acquire a preliminary idea in this aspect. However, the literature did not reveal any such study/report detailing the symptom-specific association of HFMD with seasons, as we have tried to. Some of our overall findings remain comparable with that of others5,6,9,10. Our data from the rapid appraisal of short-term surveillance demonstrated certain seasonal characteristics of local weather were associated with HFMD, like fever and rash characteristics. Moderate-to high fever (63%) was observed more often in fall/late-autumn (mid-October to November) than in early autumn (September to mid-October), yielding 37% cases (P<0.01). A similar result was obtained for papulo-vascular rashes, which predominantly occurred in fall (62%) rather than in early autumn (38%), (P<0.03).\n\nHowever, our data did not support an impact of rainfall/precipitation or ambient temperature on any of the three major signs/symptoms we have evaluated. We observed that childhood HFMD cases occurred mostly in dry weather with no rainfall (0.0 mm). Similarly, the three major symptoms of HFMD were more likely to be observed during hot/humid days, with no difference in disease severity. These findings on climatic factors or locally prevailed weather did not corroborate with the findings of others5,9,10.\n\nOne of the other unique strength of our study was to dig out an association of socio-demographic and/or HH economy of children’s families with that of childhood HFMD. The age group of victimized children (mean ± SD, 2.9± 2.3 years) remained similar to several reports1,2,7,8,14–21. However; the age of children did not differ significantly with sex. The HH structure revealed an average size of children’s family containing 5.5±0.7 persons/HH, 62% of who were the first child and 38% were the second children. The HH income scale/grades, following the World Bank standard family/HH income-groups31, highlighted that the majority of families (85%) belonged to middle-income HHs living on a modest budget. While 34.3% had upper-mid incomes, 51% had lower-mid levels of income. However, 14.7% belonged to the low-income group, who were to live on a very tight HH budgets. Notably, but logically (based on ecology, environment, health care facilities, etc.) HFMD infection was observed more among first siblings and from families significantly associated with living on tight/low HH-budget than in second sibling (P<0.01). This finding should be noteworthy as one of our unique findings associating among family size, child’s-sibs and HH-budget/family-economy. These findings might have several multifaceted reasons, but our postulation go in favor of gross inadequacy in health care expenditure by individual families, the distance of PMC-GH from respective HHs and of course, total family income as one of the major concerns. Although such socio-economic parameters and public health issues demand to be explored further, some studies highlighted that the occurrence of HFMD is associated with patients’ personal hygiene, post-defecation hand-washing, water and sanitation26. Surrounding environment, like food and drinking water18, including the contaminated sewage water27 also have a particular role in transmitting HFMD viruses amidst surrounding communities.\n\nOur clinico-epidemiological observation indicates childhood-HFMD has emerged in Bangladesh.\n\nSome outbreaks in Calcutta indicate that HFMD emerged in Bangladesh earlier.\n\nThe physicians’ rationally judged clinical suspicion (signs/symptoms) can establish a correct diagnosis.\n\nStringent differential diagnosis remain indispensable to exclude similar fever- or rash-causing illness.\n\nLaboratory diagnosis seems unessential, particularly during HFMD outbreak situations when proper laboratory-diagnosis (virus culture, serology, molecular analysis) is not readily available.\n\nWe experienced that early forecasting may aid in combating HFMD outbreaks in catchment areas to curb complications more successfully.\n\nSmall-scale/localized outbreaks can be combated utilizing existing health-care/hospital set up/facilities.\n\nNo specific treatment for HFMD exists, although supportive therapy can treat cases of HFMD in a week.\n\nIt is imperative to increase mass awareness to stop transmission of HFMD viruses (air/droplet, environment).\n\nPersonal hygiene, hand washing and a pollution-free environment are mainstays of HFMD prevention\n\n\nConclusion\n\nWe could diagnose cases of childhood HFMD successfully based on clinical signs/symptoms only and all cases recovered well within a week. Stringent differential diagnosis on similar rash and/or fever diseases/syndromes were deemed indispensable. The local climate may influence HFMD. Time consuming and costly laboratory diagnosis (virological/molecular) is not essential in resource-constrained settings, particularly during outbreak situations. No specific treatments or effective vaccinations exist for this often-underestimated disease yet. Supportive therapy and strict preventive measures is able to circumvent/destroy EV or CA viruses to combat ongoing HFMD-outbreaks/threats.\n\n\nRecommendations\n\nDevelopment of a globally representative multivalent HFMD vaccine remains necessary, particularly in countries where HFMD widespread, before it becomes pandemic. Both the government health services and meteorology departments should work together since climate is shown to be an early indicator of potential HFMD outbreaks. Our findings warrant that the countrywide public health emergency operations teams be more alert towards the effective prevention and control of HFMD in resource-constrained countries like Bangladesh. The governments of such countries should come up with a well-designed, sustainable strategic plan to combat upcoming HFMD outbreaks, in close-cooperation with national and global NGOs and UN organs to prevent its pandemic threat in the near future.\n\n\nData availability\n\nDataset 1. Complete raw data from each child assessed as part of this study. DOI: 10.5256/f1000research.15170.d21103834.\n\n\nConsent\n\nWritten informed consent was obtained from the parents/guardians of each child for the publication of this report and the images contained within it.", "appendix": "Author contributions\n\n\n\nMd. Azraf Hossain Khan is currently at Department of Dermatology and Venereology, Rajshahi Medical College Hospital, Bangladesh. Kazi Selim Anwar is currently at Faculty, Department of Infectious Diseases, School of Medicine, International University of Health & Welfare (IUHW), Japan.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank Prof. Tetsuya Matsumoto, MD, PhD, Head, Dept. of Infectious Disease, School of Medicine, International University of Health and Welfare (IUHW), Narita Campus, Japan for editing the manuscript and incorporating valuable suggestions. We thank Dr. Asadur Rahman, Dept. of Pharmacology, IUHW, for assisting in figure designing/artwork and editing some part of the manuscript. We also thank Januka Khatiwada, Dept. of Public Health, IUHW, for sorting out few technical issues with the SPSS-software. Special thank goes to the PMC-GH authority for allowing us to conduct study successfully. We remain indebted and thankful to all those parents/guardians who allowed their children to take part in the study without which this endeavor would have been in futile.\n\n\nReferences\n\nWHO: A guide to Clinical management and public health response for Hand, Foot and Mouth Disease. Section 5: Clinical Features and case management. 2011; 19. Reference Source\n\nChang PC, Chen SC, Chen KT: The Current Status of the Disease Caused by Enterovirus 71 Infections: Epidemiology, Pathogenesis, Molecular Epidemiology, and Vaccine Development. Int J Environ Res Public Health. 2016; 13(9): pii: E890. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOoi MH, Wong SC, Lewthwaite P, et al.: Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol. 2010; 9(11): 1097–105. PubMed Abstract \| Publisher Full Text\n\nAswathyraj S, Arunkumar G, Alidjinou EK, et al.: Hand, foot and mouth disease (HFMD): emerging epidemiology and the need for a vaccine strategy. Med Microbiol Immunol. 2016; 205(5): 397–407. PubMed Abstract \| Publisher Full Text\n\nWang P, Goggins WB, Chan EY: Hand, Foot and Mouth Disease in Hong Kong: A Time-Series Analysis on Its Relationship with Weather. PLoS One. 2016; 11(8): e0161006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndreoni AR, Colton AS: Coxsackievirus B5 associated with hand-foot-mouth disease in a healthy adult. JAAD Case Rep. 2017; 3(2): 165–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStock I: [Hand, foot and mouth disease--more than a harmless \"childhood disease\"]. Med Monatsschr Pharm. 2014; 37(1): 4–10; quiz 11–2. PubMed Abstract\n\nVan Pham H, Hoang TNA, Duong HT, et al.: Clinical characteristics of hand, foot and mouth disease in Daklak Province, Vietnam and associated factors of severe cases. Virusdisease. 2017; 28(4): 430–433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHii YL, Rocklöv J, Ng N: Short term effects of weather on hand, foot and mouth disease. PLoS One. 2011; 6(2): e16796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Y, Wang X, Liu Y, et al.: Detecting spatial-temporal clusters of HFMD from 2007 to 2011 in Shandong Province, China. PLoS One. 2013; 8(5): e63447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAng LW, Koh BK, Chan KP, et al.: Epidemiology and control of hand, foot and mouth disease in Singapore, 2001-2007. Ann Acad Med Singapore. 2009; 38(2): 106–12. PubMed Abstract\n\nKar BR, Dwibedi B, Kar SK: An outbreak of hand, foot and mouth disease in Bhubaneswar, Odisha. Indian Pediatr. 2013; 50(1): 139–42. PubMed Abstract\n\nSharma N, Sarkar A, Mukherjee A, et al.: Epidemic of hand, foot and mouth disease in West Bengal, India in August, 2007: a multicentric study. Indian J Dermatol. 2009; 54(1): 26–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarma N, Chakraborty S, Dutta A, et al.: Hand, Foot and Mouth Disease in West Bengal, India: A Preliminary Report on Clinicovirological Trend over 3 Successive Years (2013-2015). Indian J Dermatol. 2017; 62(5): 486–490. PubMed Abstract \| Free Full Text\n\nLi T, Yang Z, Liu X, et al.: Hand-foot-and-mouth disease epidemiological status and relationship with meteorological variables in Guangzhou, southern China, 2008-2012. Rev Rev Inst Med Trop Sao Paulo. 2014; 56(6): 533–539. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVentarola D, Bordone L, Silverberg N: Update on hand-foot-and-mouth disease. Clin Dermatol. 2015; 33(3): 340–46. PubMed Abstract \| Publisher Full Text\n\nChan JH, Law CK, Hamblion E, et al.: Best practices to prevent transmission and control outbreaks of hand, foot, and mouth disease in childcare facilities: a systemic review. Hong Kong Med J. 2017; 23(2): 177–90. PubMed Abstract \| Publisher Full Text\n\nRajtar B, Majek M, Polański L, et al.: Enteroviruses in water environment--a potential threat to public health. Ann Agric Environ Med. 2008; 15(2): 199–203. PubMed Abstract\n\nNervi SJ, Bronze MS: Hand-Foot-and-Mouth Disease (HFMD) Workup. Medscape Drugs and Dis >Infectious Dis, Updated: Jun 16, 2017. (Accessed on 09/02/2018). Reference Source\n\nCheng Q, Bai L, Zhang Y, et al.: Ambient temperature, humidity and hand, foot, and mouth disease: A systematic review and meta-analysis. Sci Total Environ. 2018; 625: 828–36. PubMed Abstract \| Publisher Full Text\n\nKumar KB, Kiran AG, Kumar BU: Hand, foot and mouth disease in children: A clinico epidemiological study. Indian J Paediatr Dermatol. 2016; 17(1): 7–12. Publisher Full Text\n\nWolff K, Johnson RA, Saavedra AP, et al.: Fitzpatrick’s color Atlas and Synopsis of Clinical Dermatology. 7th ed. Mc Graw - Hil Medical. 2013; 653–655. Reference Source\n\nSarkar PK, Sarker NK, Tayab MA: Hand, Foot and Mouth Disease (HFMD): An Update. Bangladesh J Child Health. 2016; 40(2): 115–119. Publisher Full Text\n\nBian L, Wang Y, Yao X, et al.: Coxsackievirus A6: a new emerging pathogen causing hand, foot and mouth disease outbreaks worldwide. Expert Rev Anti Infect Ther. 2015; 13(9): 1061–71. PubMed Abstract \| Publisher Full Text\n\nHand-foot-and-mouth disease: Diagnosis and treatment. Mayo Clinic. (Accessed on: 3/7/2018). Reference Source\n\nRuan F, Yang T, Ma H, et al.: Risk factors for hand, foot, and mouth disease and herpangina and the preventive effect of hand-washing. Pediatrics. 2011; 127(4): e898–904. PubMed Abstract \| Publisher Full Text\n\nConnell C, Tong HI, Wang Z, et al.: New approaches for enhanced detection of enteroviruses from Hawaiian environmental waters. PLoS One. 2012; 7(5): e32442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiao J, Yu S, Yang F, et al.: Short-Term Effects of Climatic Variables on Hand, Foot, and Mouth Disease in Mainland China, 2008–2013: A Multilevel Spatial Poisson Regression Model Accounting for overdispersion. PLoS One. 2016; 11(1): e0147054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuenpa J, Chieochansin T, Linsuwanon P, et al.: Hand, foot, and mouth disease caused by coxsackievirus A6, Thailand, 2012. Emerg Infect Dis. 2013; 19(4): 641–643. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBangladesh Population Census 2001: Bangladesh Bureau of Statistics; Cultural survey report of Pabna District 2007; Cultural survey report of upazilas of Pabna District. 2007. Reference Source\n\nHow are the income group thresholds determined? World Bank Data Help Desk. 2016. Reference Source\n\nWMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. Adopted by 64th WMA General Assembly, Fortaleza, Brazil. 2013. Reference Source\n\nNATION: Firm working on vaccine to treat HFMD. 2010; (Accessed 25 April, 2018). Reference Source\n\nHossain Khan MA, Anwar KS, Muraduzzaman AKM, et al.: Dataset 1 in: Emerging Hand Foot Mouth Disease in Bangladeshi Children- First Report of Rapid Appraisal on Pocket Outbreak: Clinico-epidemiological Perspective Implicating Public Health Emergency. F1000Research. 2018. Data Source\n\nCai Y, Liu Q, Huang X, et al.: Active immunization with a Coxsackievirus A16 experimental inactivated vaccine induces neutralizing antibodies and protects mice against lethal infection. Vaccine. 2013; 31(18): 2215–2221. PubMed Abstract \| Publisher Full Text\n\nLin CW, Chang CY, Chen WL, et al.: Formulation and immunological evaluation of a trivalent vaccine comprising emulsified submicron particles and inactivated virions of H5N1/EV71/JEV. Hum Vaccin Immunother. 2013; 9(11): 2378–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCai Y, Ku Z, Liu Q, et al.: A combination vaccine comprising of inactivated enterovirus 71 and coxsackievirus A16 elicits balanced protective immunity against both viruses. Vaccine. 2014; 32(21): 2406–2412. PubMed Abstract \| Publisher Full Text\n\nKim EJ, Park HS, Yoon HS, et al.: Four cases of onychomadesis after hand-foot-mouth disease. Ann Dermatol. 2014; 26(6): 777–778. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "38927", "date": "02 Oct 2018", "name": "H Rogier van Doorn", "expertise": [ "Reviewer Expertise Clinical Microbiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the results from a prospective observational study of children attending a single hospital in Bangladesh using WHO diagnostic criteria. If this is the first time HFMD has been described from Bangladesh, this is of major relevance locally and regionally. I recommend the authors to use the STROBE guidelines/checkbox to verify whether all required data are included. The main findings are that a number of cases of HFMD among young children was described from one hospital in Bangladesh, the symptom and age distribution are relatively similar to what is known from the region. Healthcare workers should be aware of this illness, its prevention and treatment and warning signs of severe illness. I have made some comments and suggestions below, the most important being to shorten and bring more focus on the current data in the discussion.\nSpecific comments:\nAdd this sentence on the aetiology, to replace the sentence in the second paragraph of the introduction: \"HFMD is caused by several serotypes of Enterovirus A, the most common being Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) and more recently also CV-A6 and CV-A10. EV-A71 is associated with a higher proportion of severe illness.\" Please add the exact case definition that was used to enrol children. 143 children were included, how many children were eligible during the period of enrolment? How many were not enrolled because of exclusion criteria or otherwise, how many didn't consent? Can an epidemiological curve be added? Any further information on cases in the region, nearby hospitals? Were any warning signs detected during the study? Because of the epidemiology of HFMD, the preferred age stratification would be 0-6, 6-12, 12-24, 24-60 and >60 months or similar (e.g. Xing et al1) It is common to study the effects of precipitation allowing for a lag period of few days (incubation period) Reviews on the epidemiology, mortality and long-term outcome of HFMD have been published recently. These can be referenced in the discussion for clarity. The discussion deals with a broad spectrum of general topics. This is appropriate for a report to be circulated among local healthcare workers, but not for the current scientific publication. I would suggest to focus on the data from the current study for the discussion here, to broadly describe the findings and if there were any striking differences with what has been described from the region. The authors should not overinterpret the data from this relatively small sample size to look for potential associations. In the third paragraph of the discussion the authors state that outbreaks occurred in 1997 and 1998, despite forecasts. The referenced forecasts were derived from timeseries from Malaysia from 1998-2006 and could not have predicted the 1997-8 outbreaks. There are syndromic and serotype specific timeseries from Japan dating back to the 1980s that may have forecasted these, but to my knowledge no major HFMD outbreaks had occurred in Malaysia and Taiwan prior to these. In the laboratory diagnosis section, it is important to realise that diagnosis of EV-A71 as the main pathogen is important as it is associated with a higher proportion of severe illness.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4070", "date": "09 Nov 2018", "name": "Kazi Selim Anwar", "role": "Author Response", "response": "Comment specific response from the authors:The authors thankfully appreciate the reviewer-1 for approving the paper with reservations and thank for the kind review and comments. The followings remains the comment specific response from the authors:Comment-1: The authors describe results from a prospective observational study of children attending a single hospital in Bangladesh using WHO diagnostic criteria. If this is the first time HFMD has been described from Bangladesh, this is of major relevance locally and regionally.Reply-1: Yes, this observational study in our hospital remains the first report on HFMD from Bangladesh. We, therefore, thank you sincerely, for such an important comment…. “it remains of major relevance locally and regionally”. Comment-2: I recommend the authors to use STROBE guideline/checkbox to verify if all required data includedReply-2: Yes, agreed. We have uploaded STROBE guideline to verify our data in revised version. Comment-3: The main findings that a number of cases of HFMD in young children was described from one hospital in Bangladesh, symptom & age distribution are relatively similar to what is known from the region.Reply-3: Yes, we are glad to see your valuable comment that HFMD symptoms in younger children remain relatively similar with that of other reports from South/SE- Asian region. Comment-4: Healthcare workers should be aware of this illness, its prevention and treatment and warning signs of severe illnessReply-4: Yes, we appreciate your comment that healthcare workers must be aware of HFMD its prevention & treatment, particularly on warning signs of severe illness. Mentioned in 8th bullet point of “Insights on principal findings”. Comment-5: I have made some comments and suggestions below, the most important being to shorten and bring more focus on the current data in the discussion.Reply-5: Yes, agreeing to your most suggestion, shortened the discussion part focusing on current data-that really makes sense. Shortened discussion part as suggested Comment-6: Add this sentence on the aetiology, to replace the sentence in 2nd para-graph of introduction ….\"HFMD is caused by several serotypes…. with a higher proportion of severe illness.\"Reply-6: We thankfully agreed to replace it in the 2nd paragraph of introduction.. HFMD is caused by several serotypes of enterovirus A, the most common being enterovirus A71 (EV A71) and coxsackievirus A16 (CV-A16) and more recently, also (CV A-6, and CV A-10). EV-A71 is associated with a higher proportion of severe illnesses \" Comment-7. Please add the exact case definition that was used to enroll childrenReply-7: Though it is mentioned in Fig.4, we have reemphasized the WHO recommended exact case definition of HFMD: having i) fever or history of fever, ii) papulovesicular rash on hand & foot iii) with or without oral ulcers.Added this case definition in 2nd line of method and in clinical diagnostic tool, as well. Comment 8: .… 143 children were included how many were eligible during enrolment period? How many were not enrolled because of exclusion criteria or otherwise, how many didn't consent?Reply-8: Since it was an outbreak situation, we had to enroll all 143 children attending our hospital from Sept. to Nov., 2017 with suspected HFMD cases (who met WHO criteria). Guardians of all children provided written consent to enroll. Comment 9: Can an epidemiological curve be added?Reply-9: Well yes, but we have described almost all epidemiological features in tables. Comment 10: Any further information on cases in the region, nearby hospitals?Reply-10: No. We explored to determine that among surrounding families, nurseries or kindergarten/primary schools, but none revealed any positive information. Comment 11: Were any warning signs detected during the studyReply 11: No, not as such. Of the 3 complications that we observed, only girl had onychomadesis, 1 child had pneumonia and the other had pyoderma. These may well be regarded as ‘cautionary’, if not ‘warning’ signs. Comment 12: Because of the epidemiology of HFMD, the preferred age stratification would be 0-6, 6-12, 12-24, 24-60 and >60 months or similar (Xing et al1)Reply 12: Yes. But during that pocket outbreak our hospital team categorized the HFMD victimized children into two groups of <5 and >5 years only. Since 77% of them fell under <5 years it was further categorized into <3 years & 3.1 to <5 years. This age-stratification was done to fit aged-matched cases facilitating analysis. Reply 13: It is common to study the effects of precipitation allowing for a lag period of few days (incubation period)Reply 13: Yes. But we could not do that due to paying more attention in tackling/combating the on-going emergency of that pocket outbreak. Comment 14: Reviews on the epidemiology, mortality and long-term outcome of HFMD have been published recently. These can be referenced in the discussion for clarity.Reply 14: Well, yes. But we have described some of those in our discussion already. Comment 15: The discussion deals with a broad spectrum of general topics. This is appropriate for a report to be circulated among local healthcare workers, but not for the current scientific publication.I would suggest to focus on the data from the current study for the discussion here, to broadly describe the findings and if there were any striking differences with what has been described from the region. The authors should not overinterpret the data from this relatively small sample size to look for potential associations.Reply 15: Thanks for the good suggestions. We have shortened the discussion part, focused on our data from our current study and tried to describe the striking findings only that yielded some regional differences. And we also tried to avoid over-interpreting our data (relatively small sample size). Comment 16: In the third paragraph of the discussion the authors state that outbreaks occurred in 1997 and 1998, despite forecasts. The referenced forecasts were derived from time series from Malaysia from 1998-2006 and could not have predicted the 1997-8 outbreaks. There are syndromic and serotype specific time series from Japan dating back to the 1980s that may have forecasted these, but to my knowledge no major HFMD outbreaks had occurred in Malaysia & Taiwan prior to these.Reply 16: Thanks for pointing it out rightly. After cross checking on the contents of this sentence we have removed the following sentences 'Despite epidemiological forecasts that HFMD outbreaks occur in a 2–3-year cyclical pattern two large epidemics broke out in 2 consecutively years: one in Malaysia during 1997 and the other in Taiwan, the following year.\" Corrected this part as edited in the 2nd paragraph of ‘Potentials & dynamics of HFMD outbreak’. Comment 17: In the laboratory diagnosis section, it is important to realise that diagnosis of EV-A71 as the main pathogen is important as it is associated with a higher proportion of severe illness.Reply 17: Yes. Good point. We have added this point in lab diagnosis sect giving importance to diagnose EV-A71 as the main pathogen causing proportionately more severe cases of HFMD. It was reflected in 3rd paragraph of laboratory diagnosis, properly. Finally the authors thank the reviewer-1 for his kind comments and suggestions once again." } ] } ]	1	https://f1000research.com/articles/7-1156
https://f1000research.com/articles/8-979/v1	27 Jun 19	{ "type": "Case Report", "title": "Case Report: Purple urine bag syndrome in woman with neurogenic bladder", "authors": [ "Senohadi Boentoro", "Nugroho Budi Utomo", "Senohadi Boentoro" ], "abstract": "Purple urine bag syndrome (PUBS) is a rare phenomenon in patients that is associated with the use of a long-term/indwelling urinary catheter. The purple color results from indigo and indirubin, accumulated from bacteria-mediated tryptophan conversion. High risk patients include: the elderly; women; immobilized patients; patients with an indwelling catheter, chronic constipation, alkaline urine or poor hygiene; and those with catheter bags and tubes made of certain types of plastic. We reported PUBS in an elderly woman with an indwelling catheter and chronic constipation which, to our knowledge, was the first case in our hospital. The patient underwent urinary catheter change and received intravenous ciprofloxacin, following which the urine returned to a yellow color and the patient was discharged. This case report describes the diagnosis, management and also strategies for the prevention of PUBS in Gatot Soebroto Army Hospital, Indonesia.", "keywords": [ "purple urine bag syndrome", "neurogenic bladder", "elderly woman", "indwelling catheter" ], "content": "Introduction\n\nPurple urine bag syndrome (PUBS) is a clinical phenomenon associated with urinary tract infections (UTIs) due to long-term use of catheters, usually occurring in elderly patients. PUBS was first reported by Barlow and Dickson in 19781. This phenomenon can cause panic for patients and caregivers because the color changes from yellow to purple. This change is known to occur only in the urine bag, while the color of urine does not actually change to purple2,3. Hereby, we report this rare phenomenon in an elderly woman with neurogenic bladder which, to our knowledge, was the first PUBS case in Gatot Soberoto Army Hospital, Indonesia and the first published case of PUBS from Indonesia.\n\n\nCase presentation\n\nA 64-year-old retired Southeast Asian woman was admitted to Gatot Soebroto Army Hospital, Jakarta because of fever for two days and had a consultation with a urologist because the urine in her urine bag had changed color to purple (Figure 1). This discoloration was first noticed by her daughter at home approximately three hours before the hospital admission.\n\nThe patient was known to have neurogenic bladder caused by a spinal cord injury. She had undergone several rehabilitative treatment sessions and had been using a catheter for three months. The patient had also suffered from constipation for three months previously. At the consultation, no abnormalities were found in the flank region. The patient’s bladder was palpated to ensure that it was empty, and no masses or pain were found in the suprapubic region. There were no abnormalities observed upon physical examination except motor weakness (paraparesis) in both lower extremities. The patient had a 16-Fr indwelling catheter and purple urine production of 1,350 ml/24 hours.\n\nLaboratory blood tests, including complete blood count, renal function test, liver function test and electrolyte levels, showed an increase in leukocytes (12,680/μL [normal range 5,000-10,000/μL]), with other test results within normal limits. Urinalysis, including macroscopic and microscopic analysis (test for color, sedimentation, erythrocytes, urinary casts, sedimentation, epithelial cells, crystals, bacteria, specific gravity, pH, albumin, glucose, bilirubin, urobilinogen, blood, nitrite and leucocyte esterase), showed urinary alkalosis (pH 8.5 [normal range 4.5-8.0]), nitrite positive (+2), leucocyte esterase positive (+2), with other test results within normal limits. Urine culture for aerobic bacteria, anaerobic bacteria and yeast was carried out before the administration of antibiotics. Escherichia coli culture results showed significant growth (>100,000/mL after 24 hours), which was sensitive to ciprofloxacin, nitrofurantoin and amikacin.\n\nThe patient was then given intravenous ciprofloxacin (400 mg q12h) and antipyretic (paracetamol 500mg when needed). The catheter and plastic bag were changed, and changes to the urine color occurred. After the administration of ciprofloxacin therapy (400mg q12h) for two days, the fever was resolved, and the urine returned to a clear yellow color. To avoid the development of antimicrobial resistance, ciprofloxacin (400mg q12h) was continuously given for a total of seven days. The patient was discharged afterwards and there was no further follow-up. The course of illness is shown as a timeline in Figure 2.\n\n\nDiscussion\n\nAlthough UTIs can occur at various age ranges, PUBS is commonly found in the elderly, especially in women, patients with chronic catheter use, patients with chronic constipation or for UTIs associated with sulfate/phosphatase production2. The prevalence of UTIs is estimated to be around 8.3-42,1% in hospitalized patients with long-term catheter use4. Around 9.8% of hospitalized patients with long-term catheter use experience PUBS2.\n\nMost PUBS patients use long-term catheters because of disturbances to mobilization, such as patients who use wheelchairs or are confined to bed rest5. The mechanism of how constipation causes PUBS is through changes in intestinal motility. Constipation can prolong tryptophan transit, resulting in increased levels of indoxyl sulfate in the urine3,5. A short urethra in women is a UTI predisposing factor, which is also seen in PUBS patients5. Dehydration is also considered as one of the risk factors for PUBS, due to increased indigo concentration and indirubin in the urine4. In addition, other PUBS risk factors are the use of catheters made of polyvinyl chloride plastic5. There is an increased risk of PUBS in patients with renal failure, associated with a decrease in indoxyl sulfate clearance, so bacteria produce more indigo and indirubin4.\n\nThe purple etiology of PUBS comes from the mixture of red (indirubin) and blue (indigo) chemicals resulting from tryptophan metabolism. Bacteria in the intestine metabolize tryptophan, producing indoles that are then absorbed into the portal circulation. In the liver, indoxyl sulfate is produced from indole conversion. Indoxyl sulfate is digested by bacteria, which produce indoxyl sulfatase and convert it to indoxyl. Then indoxyl is excreted through urine. In alkaline urine, indoxyl changes to indirubin (red) and indigo (blue), and the mixture of these two colors produces purple (Figure 3).\n\nOther causes of purple coloration are conjugated bile acids or steroids. The conversion of tryptophan to indole increases with excessive growth of colon bacteria in patients with chronic constipation. Both this change in urine composition and chronic constipation cause the prevalence of PUBS to be higher in patients who have constipation and long-term use of urinary catheters2.\n\nSome bacteria known to be associated with PUBS are Providencia stuartii, Providencia rettgeri, Klebsiella pneumoniae, Proteus sp., Escherichia coli, Enterococcus sp., Morganella morganii, and Pseudomonas aeruginosa2. In patients with long-term catheter use, P. mirabilis is a particular problem for medical care. Urinary catheters are considered one of the main risk factors for hospital-acquired infections based on previous prevalence studies. Therefore, the unnecessary use of urinary catheters should be avoided to reduce UTI rates.\n\nManagement of PUBS consists of improvements to hygiene, including replacement of urine catheter, management of constipation and eradication of the UTI. Control and prevention of catheter-associated UTIs should be an important element of the medical care for these patients5. The most commonly given antibiotic for PUBS is ciprofloxacin (quinolone), which is considered appropriate empirical therapy4. Some other antibiotic options that can be chosen are piperacillin/tazobactam, ticarcillin/clavulanate, ampicillin/sulbactam, ceftazidime, cefepime, levofloxacin, norfloxacin, moxifloxacin, meropenem and ertapenem. These antibiotics are effective against gram-negative bacteria5. Based on suspicion, antibiotics can be started before the results of urinalysis and urine culture are available5. In immunocompromised patients and persistent PUBS, antibodies can be given intravenously4. In previous studies, PUBS was suggested to be a sign of Fournier gangrene in immunocompromised patients and so more attention is needed to these patients. Non-plastic urine bags as a prevention method for PUBS can also be taken into consideration.\n\nAs the first reported case in our hospital, we hope this study could help in avoiding the underdiagnosis of PUBS by medical staff, especially in our hospital and in Indonesia. The limitation of this study was that there was no follow-up after patient discharge.\n\n\nConclusion\n\nAlthough PUBS is not a dangerous clinical condition, the sudden discoloration of urine to purple can cause panic for patients and families. No special management for patients with PUBS is needed apart from appropriate antibiotics according to culture results, good catheter hygiene when using catheters for a long period of time, replacement of catheters and urine bags on time and treatment of constipation. This phenomenon of PUBS needs to be known, not only by urologists, but also by other doctors and medical personnel, so that panic does not arise, and personnel can help calm patients and families. In managing patients, especially elderly patients and women, with long-term catheterization and chronic constipation, we should be aware of the risk of developing UTIs and ensure good catheter hygiene to avoid any preventable complications, including PUBS.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKhan F, Chaudhry MA, Qureshi N, et al.: Purple urine bag syndrome: an alarming hue? A brief review of the literature. Int J Nephrol. 2011; 2011: 419213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl Montasir A, Al Mustaque A: Purple urine bag syndrome. J Fam Med Primary Care. 2013; 2(1): 104–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeters P, Merlo J, Beech N, et al.: The purple urine bag syndrome: a visually striking side effect of a highly alkaline urinary tract infection. Can Urol Assoc J. 2011; 5(4): 233–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalsi DS, Ward J, Lee R, et al.: Purple urine bag syndrome: a rare spot diagnosis. Dis Markers. 2017; 2017: 931872. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang HW, Su YJ: Trends in the epidemiology of purple urine bag syndrome: A systematic review. Biomed Rep. 2018; 8(3): 249–256. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "52319", "date": "23 Aug 2019", "name": "Chinmay Patel", "expertise": [ "Reviewer Expertise Nephrology", "Internal 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\nThis is a well written manuscript. The authors have nicely described the case, pathogenesis of Purple Urine Bag Syndrome (PUBS) and followed up with excellent management points. PUBS is a rare but important phenomenon seen in elderly patients with UTI and indwelling foley catheters. Such patients can be nonverbal and asymptomatic with purple urine bag being the only sign of UTI! Doctors and other health care professionals should be familiar with this phenomenon due its morbidity.\n\nI recommend this manuscript for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "52318", "date": "02 Sep 2019", "name": "Kannan Rajendran", "expertise": [ "Reviewer Expertise general medicine", "diabetology" ], "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 hospital and country name can be avoided.\n\nThe article is very well written.\n\nGood photo.\n\nDescription is clear.\n\nIt can be accepted for publication.\n\nThe editing part also acceptable.\n\nPUBS nowadays is becoming more common, it was one of the rare entities earlier. We have reported twice from our hospital.\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/8-979
https://f1000research.com/articles/7-1624/v1	10 Oct 18	{ "type": "Research Article", "title": "Large-scale analysis of B-cell epitopes of envelope: Implications for Zika vaccine and immunotherapeutic development", "authors": [ "Iman Almansour", "Rahaf Alfares", "Halah Aljofi", "Rahaf Alfares", "Halah Aljofi" ], "abstract": "Background: Cases of the re-emergence of Zika virus in 2015 were associated with severe neurologic complications, including Gillien-Barre syndrome in adults and congenital Zika syndrome in newborns. The major structural determinant of immunity to the Zika virus is the E protein. Although B-cell epitopes of Zika E protein were recently identified, data regarding epitope variations among Zika strains in pre-epidemic and epidemic periods are lacking. Methods: Here, we conducted systematic bioinformatics analyses of Zika strains isolated between 1968 and 2017. Multiple sequence alignment of E protein as well as B-cell epitopes annotations were performed. In addition, homology-based approach was utilized to construct three-dimensional structures of monomeric E glycoproteins to annotate epitope variations. Lastly, of N-glycosylation patterns and prediction of protein stability upon mutations were also investigated. Results: Our analyses indicates that epitopes recognized by human mAbs ZIKV-117, ZIKV-15, and ZIKV-119 were highly conserved, suggesting as attractive targets for the development of vaccines and immunotherapeutics directed against diverse Zika strains. In addition, the epitope recognized by ZIKV-E-2A10G6 mAb derived from immunized mice was highly conserved across Zika strains. Conclusions: Our data provide new insights regarding antigenic similarities between Zika strains circulating worldwide. These data are essential for understanding the impact of evolution on antigenic cross-reactivity between Zika lineages and strains. Further in-vitro analyses are needed to determine how mutations could impact the development of vaccines that can effectively neutralize Zika viruses.", "keywords": [ "Antibody", "Bioinformatics", "Envelope", "Epitope", "Homology Modeling", "Neutralizing Antibodies", "Vaccine", "Zika" ], "content": "Introduction\n\nZika is a positive-sense, enveloped, RNA virus of the Flaviviridae family1, which also includes dengue virus, West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and yellow fever virus (YFV)2. Zika was originally discovered in a rhesus monkey in 1947 in Uganda3, and the first case of spread to humans was reported in 19524. Since that time, the virus has spread globally, with Zika outbreaks reported in Micronesia in 2007 and in the Pacific islands in 2013–20145,6. A recent outbreak that began in Brazil in 2015 eventually spread to countries in North America and the Caribbean7,8.\n\nThe Zika virus genome encodes three structural proteins (capsid [C], premembrane [PrM], and envelope [E]) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)2. Similar to other flaviviruses, the structural proteins and viral genome form virions that assembles as immature particles at the endoplasmic reticulum of infected cells9. The immature particles are composed of 60 PrM/E protein heterodimers that protrude from the viral surface10. In the Golgi apparatus, PrM is cleaved by furin-like protease2. After maturation, pr is released from the host cell, and 90 E protein homodimers rearrange in an antiparallel orientation to form a herringbone-like array11,12.\n\nThe E protein is the major surface glycoprotein of flaviviruses and plays an essential role in virus attachment and fusion. Each E protein monomer consists of three domains: DI, DII, and DIII13, which undergo major rearrangements during the virus maturation cycle14,15. DI is a central beta-barrel domain; DII is a finger-like dimerization domain; and DIII is an immunoglobulin-like domain10,11. DI, which connects DII to DIII, is essential for the conformational changes required for viral entry into cells16. DII contains a fusion loop (FL) that interacts with the endosomal membrane, whereas DIII contains the receptor-binding site and is thus essential for attachment of virus particles to the host cell15,16. DIII also plays an essential role in mediating the fusion of virus particles with the endosomal membrane after endocytosis17.\n\nThe global spread of Zika virus in conjunction with the neurologic consequences of infection have increased the urgency of efforts to develop Zika vaccines and immunotherapeutics. Humoral immunity is the major source of host protection against flaviviruses, in which neutralizing antibodies play an important role in virus clearance18. Antibodies generated against the E protein have been shown to block the entry of viruses into host cells19. Previous attempts to map the antigenic epitopes of the Zika E protein utilized antibodies specific for other flaviviruses, such as dengue virus20–22. Recently, several B-cell antigenic epitopes within an individual E domain were identified in studies of antibodies isolated from Zika-virus infected patients23–25 and Zika-vaccinated mice13,26,27.\n\nAlthough antigenic epitopes of the E protein have been characterized based on maps prepared using Zika-specific antibodies, no systematic analyses of the specificity of Zika monoclonal antibodies (mAbs) for available Zika E protein sequences have been conducted. Such data are particularly important, as RNA viruses exhibit high mutation rates and can generate mutations that enable them to evade the host immune system. Importantly, the structural stability of E protein is a key factor for antibody binding. The amino acids substitutions and subsequent effects on structural stability and antibody binding can be performed by a homology based in-silico approach of the three-dimensional (3D) structure of E proteins. Hence, analyzing sequence data to annotate mutations at key residues and subsequent prediction of the effect of those mutations on protein structure stability is essential. Therefore, identifying novel amino acids mutations that are likely to contribute in the immune evasion is important.\n\nIn the present study, therefore, we extracted all of the available E protein sequences for Zika isolates obtained from 1968 to 2017 and constructed three-dimensional (3D) structures of E proteins from various Zika strains using homology modeling. We also investigated the patterns and conservation of E protein B-cell epitopes and assessed their structural stability upon mutation.\n\n\nMethods\n\nComplete Zika polypeptide sequences for isolates identified between 1968 and 2017 were obtained from the National Center for Biotechnology Information (NCBI) Zika resource28. A total of 409 complete polypeptide sequences were retrieved, and duplicate sequences were removed. Multiple sequence alignment was performed using MUSCLE in the Geneious tool, version 11.0, and the E protein region as extracted29. Lastly, MUSCLE alignment was performed for E protein sequences and duplicate E sequences were subsequently removed.\n\nA phylogenetic tree for all unique Zika virus E protein sequences was constructed using the maximum-likelihood method with the PhyML tool, version 3.030, with 100 bootstrap replications. The tree applied an LG substitutional model to determine the divergence of E protein sequences. Lastly, the phylogenetic tree was edited using the Figtree tool, version 1.4.3.\n\nThe SWISS model31 server was used to generate 3D structures of the E proteins of Zika isolates identified in 1968, 2007, 2013, 2015, and 2016. Chain A of the E protein structure (PDB: 5GZN) was used as a template for homology modeling. The best homology model was selected based on global model quality estimate (GMQE) and Qmean statistics. Each homologous 3D structure was evaluated using Ramachandran plots prepared with PROCHECK32. Hydrogen bonds were added using molprobity33. Each model was subjected to energy minimization using the ModRefiner server described by Xu and Zhang34.\n\nE protein-specific antigenic epitopes of monoclonal antibodies (mAbs) isolated from Zika-virus infected humans and Zika immunized mice were retrieved from the Immune Epitope Database (IEDB)35, which is a free resource funded by the National Institute of Allergy and Infectious Diseases devoted to disseminating antigenic epitope data. Linear and conformational B-cell epitopes with positive major histocompatibility complex ligands were selected. B-cell epitope regions mapped with B-cell receptor (BCR)-positive neutralizing antibodies were also selected. Epitopes that mapped with screening peptides and did not elicit an immune response were removed. A total of 7 human and 10 mouse (from mice immunized with E protein) B-cell epitopes were identified. The identified epitopes were annotated against aligned E sequences as well as the 3D structures of monomeric E proteins using the Chimera tool36. Potential sites of N-glycosylation were predicted using NetNglycan 1.0 server37. Potential N-glycosylation sites were defined by the sequence Asp/X/Ser/Thr, where X represents any amino acid except Pro. A threshold of >0.5 suggested an N-glycosylated residue.\n\nThe effect of mutations on the stability of E protein was predicted using the mutation cutoff scanning matrix (mCSM)38, site-directed mutator (SDM)39, DUET40, and I-Mutant 2.041 tools. The mCSM is a machine-learning algorithm based on a 3D physiochemical environment, and the data are summarized as a graphical signature. The SDM is a statistical potential energy function based on the propensity of amino acids in wild-type and mutant proteins to assume folded and unfolded conformations. DUET is an integrated computational approach that utilizes both SDM and mCSM to predict the effect of non-synonymous single-nucleotide polymorphisms on protein stability. Lastly, the I-Mutant webserver is a neural network-based tool for predicting mutation-associated free energy changes. The I-Mutant2.0 tool enables prediction of free energy changes under differing conditions of pH, temperature, neighboring residues, and solvent accessibility.\n\n\nResults\n\nAntigenic variations among Zika strains were examined by first obtaining the complete sequences of Zika polypeptides from the NCBI Zika resource19. A total of 409 Zika polypeptide sequences were retrieved. Identical polypeptide sequences were removed, resulting in a final total of 257 sequences. Sequences were aligned by MUSCLE using Geneious software, E protein sequences were extracted, and duplicate E protein sequences were removed, resulting in a total of 75 unique sequences (Table 1). Of note, the majority of the 75 unique E protein sequences were represented strains isolated in 2015 and 2016. Sequences from isolates collected in 2017 did not harbor any unique mutations in comparison to sequences from isolates collected in previous years; thus, 2017 sequences were removed after duplicate E protein sequences were removed.\n\nA phylogenetic tree of unique E protein sequences was constructed using the maximum-likelihood function in PhyML software, with 100 bootstrap replications (Figure 1). Zika strain accession numbers shown in the phylogenetic tree denote the year of isolation. Notably, E protein sequences from isolates collected in 1968, 2007, 2010, and 2012 clustered in one group, indicating that the associated strains are closely related (Figure 1). However, sequences from strains isolated in 2013 and 2014 exhibited divergence from the sequences of strains isolated in previous years (Figure 1).\n\nYear 1968 colored (purple), 2007 (yellow), 2010 (cyan), 2012 (green), 2013(red), 2014 (brown), 2015(magenta), and 2016 (blue).\n\nA number of recent reports describe the isolation of mAbs specific for Zika E protein23–28. These antibodies bind preferentially to epitopes located in DII and DIII of the E glycoprotein (Table 2). A total of 7 neutralizing mAbs have been isolated from Zika-virus infected humans, and all of these mAbs bind to discontinuous epitopes of E protein. Of note, ZIKV-117 and ZIKV-19 mAbs recognize epitopes located in DII (Table 2), whereas ZIKV-12 and ZIKV-15 mAbs recognize epitopes located in the FL region, and mAbs ZIKV-Z006, and ZIKV-116 as well as the ZKA 190 mAb recognize epitopes located in DIII. Significant overlap between antigenic epitopes specific for ZIKV-Z006 and ZIKV-116 was observed, as three residues in the epitope recognized by the ZIKV-116 mAb are shared with the epitope recognized by ZIKV-Z006 (Table 2). In addition, the epitope region recognized by the ZKA 190 mAb overlapped with that of the mAb specific for ZIKV-Z006 in two amino acids residues.\n\nA total of 10 B-cell epitopes of Zika E protein were found to elicit humoral antibody responses in vaccinated mice (Table 2). Five of these epitopes were shown to be linear and elicited the production of neutralizing antibodies (Table 2). An additional five discontinuous epitopes have been characterized based on antibodies obtained from vaccinated mice (Table 2). The majority of those epitopes are bound to DIII domain of E.\n\nTo identify amino acid substitution mutations occurring in B-cell epitopes, we aligned the sequences of 75 unique E protein amino acids sequences among the 422 pre-epidemic and epidemic Zika strains identified. The sequence of Zika/Nigeria/9/9/1968 was used as a reference, and the E protein sequences were mapped against all of the mAbs from Zika infected humans or vaccinated mice. We then annotated the mutations in Zika E glycoprotein at predefined B-cell epitopes. Only nine Zika strains were found to carry mutations in the predefined B-cell epitopes recognized by mAbs from Zika-virus infected humans. Of note, two amino acids substitutions (R335T) and (D393E) appeared in 2007 (Figure 2A). These amino acids substitutions were retained in all subsequent strains from 2007 to 2016. Interestingly, no unique mutations were observed in predefined B-cell epitopes for isolates collected between 2008 and 2014. However, in 2015, two additional substitutions of alanine residues for threonine residues appeared at amino acid positions 309 and 333 (Figure 2A). These mutations were not retained in subsequently isolated Zika strains, with the sequences quickly reverting to those of previously isolated strains. The greatest number of amino acid substitution mutations occurred in 2016; the majority of the mutations identified in 2016 involved several deletions in specific regions of the predefined B-cell epitopes (Figure 2A). Of note, the Dominican Republic/6/6/2016 strain exhibited significant deletions in B-cell epitopes recognized by ZIKV-Z006 mAb (Figure 2A). Surprisingly, 3 B-cell epitopes were completely conserved in the pre-epidemic and epidemic strains and indeed have not changed for nearly 50 years (Figure 2A). These epitopes are recognized by the mAbs ZIKV-117, ZIKV-15, and ZIKV-19. Thus, as these mAbs could exhibit cross-reactivity against a wide range of Zika strains, they have potential for use in the development of vaccines and immunotherapeutics targeting Zika (Figure 2A).\n\nA total of 9 Zika strains are variable at predefined B-cell epitopes during 1968–2017.\n\nConversely, none of 10 mouse B-cell epitopes were completely conserved among all Zika strains (Figure 2B, Figure 2C). Of note, the number of Zika strains exhibiting variations in the amino acid sequence at predefined antigenic epitopes of the E protein was higher in mAbs characterized in vaccinated mice than in mAbs characterized in infected humans (Figure 2B, Figure 2C). In addition, both the linear and discontinuous epitopes of vaccinated mice exhibited variations beginning in 2014 and continuing in subsequent years (Figure 2B, Figure 2C). The majority of mAbs in vaccinated mice recognized the DIII of E protein, and these epitopes exhibited higher rates of sequence variation than did the mAbs recognizing DII (Figure 2B, Figure 2C). Of note, the ZIKV-E-2A10G6 mAb binds to a highly conserved discontinuous epitope in which a single amino acid deletion was observed in Zika strain ATG29285\|Homo sapiens\|Mexico\|17/05/2016. Remarkably, discontinuous epitopes bound the ZV-67 mAb exhibited the highest degree of variation in amino acid sequence among all the Zika B-cell epitopes examined (Figure 2B). Sequence variations were also observed in all of the Zika E protein linear epitopes (Figure 2C).\n\nA total of 18 Zika strains were variable at predefined B-cell epitopes during 1968–2017.\n\nA total of 20 Zika strains were variable at predefined B-cell epitopes during 1968–2017.\n\nThe recently solved E protein structure of Zika enabled us to construct a homology model of various E protein sequences. Amino acid substitutions in B-cell epitopes were also annotated on the 3D structure of the E protein. The majority of mutations in the E protein were found to be located within DIII, whereas the B-cell epitopes in DII were highly conserved (Figure 3). Zika strain Homo sapiens/French Polynesia/11/13 did not harbor any additional mutations in B-cell epitopes compared with Zika strain Homo sapiens/Micronesia/01/06/07 (Supplementary Figure 1).\n\nHomology models of E were built from PDB: 5GZN chain A from strains of following E sequences: AMR68906\| Homo sapiens \|Nigeria\|09/09/1968, ACD75819\| Homo sapiens \|Micronesia\|01/06/2007, ANO46307\| Homo sapiens/French Polynesia/11/2013, AMX81917/Homo sapiens/Thailand/16/01/2015, and AMQ48986 \| Homo sapiens \|USA\|02/02/2016. Color blue color represents amino acids conservation at B-cell epitopes and color pink represents amino acids substitutions. Yellow label represent N-glycosylation potential of E.\n\nIt is known that N-glycosylation can mask antigenic epitopes. However, as the glycosylation site in the Zika E protein is located remotely from the predefined B-cell epitopes, glycosylation does not mask the B-cell epitope epitopes (Figure 3). This is consistent with reports of glycosylation in E protein of WNV and JEV18. A recent report demonstrated that glycosylation at 154 is critical for Zika infection of both mammalian and mosquito hosts17. Our analyses indicate that antigenic changes occur less frequently in Zika strains (Figure 3) and suggest that highly effective neutralizing Zika vaccines and immunotherapies for treating infections with known Zika strains are possible. Consequently, monitoring antigenic changes in E proteins over time would be useful for evaluating the cross-neutralizing potential of Zika vaccines against newly mutated strains.\n\nMutation stability was carried out to predict the effects of non-synonymous variants on the stability of E protein and antibody binding. Here, we analyzed the stability of monomeric E protein upon substitutional mutations. To investigate the effect of amino acids substitutions at antigenic epitopes on the stability of E protein and antibody binding of E, 4 prediction tools for mutational stability were selected.\n\nWe predicted the stability of B-cell epitope mutations using the 3D structure of Zika E proteins. In both the T309A and T335A substitutions, a polar threonine residue was substituted with a hydrophobic alanine residue. No change in hydrophobicity with the V391I mutation or change in charge with the D393E were observed. In the R335T mutation, a basic residue was substituted with an aromatic residue. Overall, these suggest that defined substitutions in the E glycoprotein are potentially destabilizing. However, these mutations had moderate destabilizing effect, as the ∆∆G values ranged between -0.3 and -0.7 kcal/mol (Table 3).\n\n\nDiscussion\n\nAttempts to control the spread of Zika virus via mosquito control have met with limited success. Indeed, within the past 3 years, a Zika pandemic occurred. There is a significant gap in knowledge regarding immunogenic cross-reactivity between Zika strains, even six decades after the first human infection was reported. Bioinformatics approaches can play vital roles in identifying rapidly evolving amino acid residues and thereby facilitate precise mapping of key residues that drive antigenic escape in response to the generation of host neutralizing antibodies. In the present study, we evaluated conserved versus rapidly evolving antigenic regions in predefined B-cell epitopes of the Zika E protein in pre-epidemic and epidemic periods.\n\nThe finger-like DII of the Zika E protein contains a FL that is inserted into the endosomal membrane as a result of pH-dependent conformational changes14. The FL is located within the beta-sheet structure in the terminal region of DII and contains a highly conserved hydrophobic peptide that triggers the structural changes required for fusion processes under conditions of low pH15. Our analysis demonstrated that B-cell epitopes in DII of Zika E protein are highly conserved. mAbs ZIKV-117, ZIKV-15, and ZIKV-19 are bound to the highly conserved region of DII and are therefore attractive candidates in the design of Zika vaccines and immunotherapeutics.\n\nThe immunoglobulin-like DIII of the Zika E protein contains receptor-binding sites and plays an essential role in attachment and fusion of the virus to host cells11,12. Importantly, DIII reportedly induces the production of type-specific neutralizing antibodies26, as mAbs isolated from patients infected with either Zika or dengue are highly specific. In the present study, we found that epitopes within E protein DIII vary greatly within Zika strains.\n\nWhile dengue is considered as a single serotype, it is characterized by four distinct serotypes. The antibody-dependent enhancement (ADE) hypothesis holds that cross-reactive antibodies generated as a result of previous infections with heterologous flaviviruses can enhance the infectivity of other viruses42,43. Infection with the same serotype elicits a protective immune response, but re-infection with a different serotype can lead to serious disease42,43. Previous studies demonstrated that mAbs isolated from patients infected with dengue virus cross-react with Zika virus44–47.\n\nSimilarly, mAbs isolated from Zika patients directed against E protein DII cross-react with dengue44,48, indicating possible increased risk of ADE. Furthermore, another study demonstrated that dengue-virus derived mAbs also cross-react with Zika with high potency20. Those mAbs are bound to quaternary epitopes, which include the site of interaction of E protein dimer with PrM during virus maturation20. In contrast, most mAbs directed against DIII of Zika E protein do not cross-react with dengue44.\n\nIn the present study, we compared mAbs of Zika E protein elicited in cases of Zika-virus infected humans versus mAbs induced by Zika vaccination in mice and identified several conserved epitope footprints. The conserved E protein epitopes could be useful in research aimed at developing vaccines that elicit the production of antibodies that provide protection against Zika strains but do not cross-react with dengue. For example, immunization with a peptide cocktail of antigenic DIII epitopes might provide broad protection against a variety of Zika strains yet demonstrate no cross-reactivity with dengue, thus eliminating the possibility of ADE associated with the anti-Zika antibodies.\n\n\nData availability\n\nZika protein sequences data can be found at the NCBI Zika resource.\n\nZika B-cell antigenic epitopes can be found at Immune Epitope Database (IEDB).", "appendix": "Grant information\n\nThe research was supported by Deanship of Scientific Research at Imam Abdulrahman Bin Faisal University [DSR 2017-376-IRMC].\n\nThe funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgment\n\nWe would like to thank Arwa Alghamdi from College of Pharmacy at IAU who assisted in the research.\n\n\nSupplementary material\n\nHomology model for sequences of \|Nigeria\|09/09/1968, ACD75819\| Homo sapiens \|Micronesia\|01/06/2007, ANO46307\| Homo sapiens/French Polynesia/11/2013, AMX81917/Homo sapiens/Thailand/16/01/2015, and AMQ48986 \| Homo sapiens \|USA\|02/02/2016 were verified.\n\n\nReferences\n\nKuno G, Chang GJ: Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu D, Zhang Y: Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization. Biophys J. 2011; 101(10): 2525–2534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVita R, Overton JA, Greenbaum JA, et al.: The immune epitope database (IEDB) 3.0. Nucleic Acids Res. 2015; 43(Database issue): D405–D412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605–1612. PubMed Abstract \| Publisher Full Text\n\nGupta R, Jung E, Brunak S: Prediction of N-Glycosylation Sites in Human Proteins. 2004; 46: 203–206.\n\nPires DE, Ascher DB, Blundell TL: mCSM: predicting the effects of mutations in proteins using graph-based signatures. Bioinformatics. 2014; 30(3): 335–342. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorth CL, Preissner R, Blundell TL: SDM--a server for predicting effects of mutations on protein stability and malfunction. Nucleic Acids Res. 2011; 39(Web Server issue): W215–W222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPires DE, Ascher DB, Blundell TL: DUET: a server for predicting effects of mutations on protein stability using an integrated computational approach. Nucleic Acids Res. 2014; 42(Web Server issue): W314–W319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCapriotti E, Fariselli P, Casadio R: I-Mutant2.0: predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Res. 2005; 33(Web Server issue): W306–W310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKliks SC, Nisalak A, Brandt WE, et al.: Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg. 1989; 40(4): 444–451. PubMed Abstract \| Publisher Full Text\n\nLobigs M, Diamond MS: Feasibility of cross-protective vaccination against flaviviruses of the Japanese encephalitis serocomplex. Expert Rev Vaccines. 2012; 11(2): 177–187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStettler K, Beltramello M, Espinosa DA, et al.: Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353(6301): 823–826. PubMed Abstract \| Publisher Full Text\n\nPriyamvada L, Quicke KM, Hudson WH, et al.: Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proc Natl Acad Sci U S A. 2016; 113(28): 7852–7857. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwanstrom JA, Plante JA, Plante KS, et al.: Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus. mBio. 2016; 7(4): pii: e01123-16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDejnirattisai W, Supasa P, Wongwiwat W, et al.: Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat Immunol. 2016; 17(9): 1102–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKawiecki AB, Christofferson RC: Zika Virus-Induced Antibody Response Enhances Dengue Virus Serotype 2 Replication In Vitro. J Infect Dis. 2016; 214(9): 1357–1360. PubMed Abstract \| Publisher Full Text" }	[ { "id": "39301", "date": "06 Nov 2018", "name": "Meirong Jia", "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\nSummary: Zika virus is still a big threat to human health, and E protein is its major structural determinant of immunity. The identification of B-cell epitopes of Zika E protein is crucial for the development of vaccines and immunotherapeutics. Here, the authors conducted systematic bioinformatics analyses of Zika strains isolated between 1968 and 2017, they’ve found conservative epitopes between Zika strains, which could be attractive targets for treating against diverse Zika strains. The research report is generally sound and the finding is scientifically valuable, which would provide guidance for later research in Zika vaccine development. Thus, I would recommend the indexing of this work in F1000Research.\n\nThe followings are some suggestions and comments that might help to make the report more suitable for indexing:\nIn the ‘Introduction’ part, for the 2nd paragraph, “After maturation, pr is released from the host cell” is that “PrM” being released instead? In ‘Table 2’, the first row, should “mAB” be “mAb”? In the ‘Abstract’, for the ‘Results’ part, “Our analyses indicate that epitopes recognized by human mAbsZIKV-117, ZIKV-15, and ZIKV-119 were highly conserved”. Should be “ZIKV-19”, please correct it. Also, since there is mutation as shown in ‘Fig 2B’ other than the strict conservation pattern in ‘Fig 2A’, please rewrite the claim that “ZIKV-E-2A10G6 mAb derived from immunized mice was highly conserved across Zika strains”. In the ‘Results’, for E protein homology modelling, the 2nd paragraph, “Glycosylation does not mask the B-cell epitope epitopes” should be “antigenic epitopes?” In the ‘Method’ part, for ‘Mapping of antigenic epitopes’, “Potential N-glycosylation sites were defined by the sequence Asp/X/Ser/Thr, where X represents any amino acid except Pro. A threshold of >0.5 suggested an N-glycosylated residue.” Please further explain what values >0.5 would suggest an N-glycosylated residue. The authors described in the ‘Methods’ that “The I-Mutant2.0 tool enables prediction of free energy changes under differing conditions of pH, temperature, neighboring residues, and solvent accessibility.” For the data shown in ‘Table 3’, what conditions did authors apply to conduct the prediction? In the ‘Results’, the last paragraph, “Overall, these suggest that defined substitutions in the E glycoprotein are potentially destabilizing.” Could the authors further discuss what the consequence or effects of destabilization has on virus infection since authors said that the structural stability of E protein is a key factor for antibody binding. In the ‘Discussion’ part, the authors claimed that “The conserved E protein epitopes could be useful in research aimed at developing vaccines that elicit the production of antibodies that provide protection against Zika strains but do not cross-react with dengue.” Yet from the manuscript, it seems that no conserved epitopes in DIII has been identified. If so, authors might need to rewrite the last part of 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4694", "date": "27 Jun 2019", "name": "Iman Almansour", "role": "Author Response", "response": "We thank Dr.Jia for the careful reading of the manuscript and the constructive remarks. Response to general comments: 1.Corrections has been made. 2.Corrections has been made. 3.Yes, thank you for pointing this out. Corrections were made. 4.Yes, we are sorry for the unintended mistake. Corrections were made. 5. Explanation has been made. 6.pH 7.0 and temperature at 25 C were applied. The details were added in the method section. 7.In the revised document, we further explained the effect of mutations on protein stability and protein binding (Results section, mutation stability, first paragraph). 8.In the discussion section, we have explained that immunization with a cocktail of antigenic DIII epitopes might provide broad protection against a variety of Zika strains." } ] }, { "id": "42684", "date": "21 May 2019", "name": "Sunil K Joshi", "expertise": [ "Reviewer Expertise Innate Immunity", "Infectious diseases", "Vaccinology" ], "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\nAuthors described in-silico methods to delineate the data which are particularly important due to high mutation rates by RNA viruses which can generate mutations, that enable them to evade the host immune system. Authors emphasized the structural stability of E protein, which is a key factor for antibody binding. The bioinformatic analysis of B cell epitopes of Zika is very important study in terms of developing effective vaccine and may be also in cross-protection against other similar viruses. In the present study, the authors extracted all of the available E protein sequences for Zika isolates obtained from 1968 to 2017 and constructed three-dimensional (3D) structures of E proteins from various Zika strains using homology modelling. Further the authors investigated the patterns and conservation of E protein B-cell epitopes and assessed their structural stability upon mutation. The authors did meticulous data analysis and discussed very thoroughly.\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/7-1624
https://f1000research.com/articles/8-232/v1	28 Feb 19	{ "type": "Software Tool Article", "title": "Visualization of the small RNA transcriptome using seqclusterViz", "authors": [ "Lorena Pantano", "Francisco Pantano", "Eulalia Marti", "Shannan Ho Sui", "Francisco Pantano", "Eulalia Marti", "Shannan Ho Sui" ], "abstract": "The study of small RNAs provides us with a deeper understanding of the complexity of gene regulation within cells. Of the different types of small RNAs, the most important in mammals are miRNA, tRNA fragments and piRNAs. Using small RNA-seq analysis, we can study all small RNA types simultaneously, with the potential to detect novel small RNA types. We describe SeqclusterViz, an interactive HTML-javascript webpage for visualizing small noncoding RNAs (small RNAs) detected by Seqcluster. The SeqclusterViz tool allows users to visualize known and novel small RNA types in model or non-model organisms, and to select small RNA candidates for further validation. SeqclusterViz is divided into three panels: i) query-ready tables showing detected small RNA clusters and their genomic locations, ii) the expression profile over the precursor for all the samples together with RNA secondary structures, and iii) the mostly highly expressed sequences. Here, we show the capabilities of the visualization tool and its validation using human brain samples from patients with Parkinson’s disease .", "keywords": [ "small RNA", "miRNA", "tRNA", "snoRNA", "sequencing", "visualization", "report" ], "content": "Introduction\n\nSmall RNAs are 18-36-nt-long RNA molecules that are involved in gene regulation, chromatin structure, and transposon element repression. The most well known small RNAs are miRNAs, endo-siRNAs and piRNAs1. They are typically processed from double-stranded RNA molecules or single-stranded RNA molecules with a hairpin structure2. They bind to members of the Argonaute (AGO) protein family to form the RNA-induced silencing complex that regulates other RNA molecules and plays a key role in gene silencing3,4. Small RNAs can also regulate chromatin states through histone modification and methylation5,6. Next generation sequencing technologies have enabled a deeper understanding of miRNAs, and other small RNA types have been detected. For instance, it is now known that miRNA genes generate several mature variants called isomiRs that have been detected in multiple conditions, tissues and species7. Other small RNAs can arise from mature tRNAs (tRNA fragments) or small nucleolar RNAs8,9. While the biogenesis of these molecules is not well understood, studies suggest that they bind to AGO proteins and perform similar functions10,11.\n\nHigh-throughput sequencing is a powerful technique for detecting and quantifying small RNAs. The analysis of small RNA data involves multiple steps for detection, annotation, quantification, and de novo discovery of putative small RNA molecules. In general, tools focus on the annotation of known miRNAs12, but new methods to detect other functional types of small RNAs are becoming increasingly important to understand the complex roles of small RNAs. Some tools have been developed to address this challenge13–15 but few of them produce a visual and interactive report16,17, and many depend on the use of a remote web server18–21.\n\nWe previously developed seqcluster, a genome-wide small RNA characterization tool that detects units of transcripts (clusters) using a heuristic iterative algorithm to deal with multi-mapped events22. It quantifies all types of small RNAs in non-redundant manner, and extracts patterns of expression in biologically defined groups. This allows us to study any small RNA cluster detected in the samples, including novel regions not previously discovered or small RNAs in species with poorly curated annotations. Here we describe seqclusterViz23, an interactive web-app that reports the output of seqcluster, visualizing small RNA biological features to better understand their putative functions. It allows the user to browse lists of detected small RNAs, shows the precursor secondary structures and the small RNA expression on the precursor, allowing for more in-depth characterization of isomiRs, tRNA fragments, and any other small RNAs detected.\n\nseqcluster and seqclusterViz are integrated into bcbio-nextgen, a community-based Python framework for fully automated high throughput sequencing analysis.\n\n\nMethods\n\nseqclusterViz23 is developed in HTML, CSS and JavaScript programming languages. It is a stand-alone tool without external dependencies. It runs locally on one’s computer making it portable and independent. It uses an SQLite JavaScript library to load all the information from a file created by the seqcluster tool22.\n\nseqclusterViz23 works on Opera >44.0, Firefox >52.0 and Chrome >57.0. It requires a seqcluster report as input. An Internet connection is not required. The tool can be downloaded from its home page (https://github.com/lpantano/seqclusterViz/archive/master.zip). After extracting the ZIP file content, the user can open the index.html file with the desired web browser. The user first clicks the ’UPLOAD’ button and then selects the seqcluster.db file. Once the data has been uploaded, the top-left panel displays all of the small RNA transcripts detected. Each small RNA transcript is clickable to obtain more information (1A). After selecting a small RNA transcript, the top-right panel shows the genomic locations for that transcript. The middle-left panel displays the abundance profile along the precursor (1B); the middle-right displays the RNA secondary structure (1B); and the bottom table shows the top 50 most abundant sequences. This table can be sorted and searched using text queries (1C).\n\n(A) Top panel with table showing the list of small RNAs detected (left) and genomic location (right). (B) Middle panel shows abundance profile over the precursor (left), and secondary structure (right). This is an example of batch effect at the 3’ end (blue higher than brown) and disease effect at the 5’ end (solid lines higher than dashed lines). (C) Bottom panel shows a table with the top most expressed sequences on the selected small RNA transcript. The index column is the sequence identifier that links the results to the original seqcluster output files.\n\nThe tool provides a number of formatting options to emphasize differences between groups and/or samples and to customize figures. Figures can be exported by right-clicking on it. This provides an easy and quick option to generate publication-ready material.\n\n\nUse cases\n\nWe used public data from 14 human brain samples at pre-motor (PT) and motor (CT) stages of Parkinson’s disease (GEO accession number GSE97285) and 14 healthy human brain samples (pre-motor controls - PC and motor stages control - CC)22. Data was analyzed with bcbio-nextgen using piDNA to detect the adapter24, cutadapt to remove it25, STAR to align against the hg19 genome assembly26, and seqcluster to detect small RNA transcripts22. We used the output seqcluster.db from seqcluster report command to test seqclusterViz23. It took four seconds to upload this 28 MB file to the web page. This dataset is affected by a batch effect for the two Parkinson’s groups due to the groups being sequenced at different read lengths. PC and CC samples were derived from the same RNA extraction, and were expected to show similar expression profiles. However, there is a clear difference by batch (brown versus blue) that is visually apparent in the abundance profile of the tRNA-Arg-TCT RNA across the length of the transcript in (1B). Longer reads allow for detection of longer small RNAs since the 3’ adapter can be recognized during the analysis (there is a requirement to include adapter sequences in the seqcluster tool). The longer reads from the PC/PT samples (blue) permitted detection of longer small RNAs at the end of the precursor, generating the batch difference in the abundance profile. Moreover, there is a difference in expression at the 5’ end of the precursor, where Parkinson’s samples (solid lines) are higher than their respective controls (dashed lines). The secondary structure of this small RNA shows a pre-miRNA-like hairpin structure (with a stem-bulge-stem and a terminal-loop) that is normally required to be processed into 18-33-nt mature molecules, where the stem-bulge-stem section encodes the mature sequence27,28. Although the structure is larger than typical pre-miRNAs, it is still possible to process with the miRNA machinery. Thus the secondary structure of the molecule can serve as an additional feature to evaluate when seeking candidates for further experimental validation. Quantitative polymerase chain reaction (qPCR) or small RNA transfection technologies are often used to validate small RNA stability and function. To do so, a single small RNA needs to be used as the target sequence for these assays. The table at the bottom of the page (1C) allows users to select the most abundant sequence in the current small RNA that can be used for such experiments.\n\n\nSummary\n\nseqclusterViz23 helps users to explore the expression profiles of detected small RNAs across the length of the precursor, the secondary structure of the small RNA, and the annotation. We show the importance of visualizing small RNAseq data to prioritize candidate small RNAs for further experimental validation or functional analysis. The user can modify the figure format and export it for publication or presentation purposes. It is also possible to select the most highly expressed sequence of a transcript cluster that can be used for qPCR or for cell transfection assays.\n\n\nData availability\n\nData to reproduce this analysis is available from the Parkinson project page.\n\nData from 14 healthy human brain samples were originally reported by Pantano et al.22. Data from 14 human brain samples at pre-motor (PT) and motor (CT) stages of Parkinson’s disease are available at GEO, accession number GSE97285.\n\nThe web-tool can be tested at GitHub pages. Click on Load Example to start using the tool with the example data set.\n\n\nSoftware availability\n\nseqclusterViz is downloaded from: https://github.com/lpantano/seqclusterViz/archive/v0.1.1.zip.\n\nSource code available from: https://github.com/lpantano/seqclusterViz.\n\nLink to source code as at time of publication: url https://doi.org/10.5281/zenodo.255677423.\n\nLicense: MIT License.", "appendix": "Grant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors would like to thank researchers who helped to improve this tool: Aron Gyuris, Mira Pavkovic, Maria Mavrikaki. Thank you also to Amanda King for edits.\n\n\nReferences\n\nMartens-Uzunova ES, Olvedy M, Jenster G: Beyond microRNA--novel RNAs derived from small non-coding RNA and their implication in cancer. Cancer Lett. 2013; 340(2): 201–211. PubMed Abstract \| Publisher Full Text\n\nKim VN, Han J, Siomi MC: Biogenesis of small RNAs in animals. 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PubMed Abstract \| Publisher Full Text\n\nYang JH, Qu LH: DeepBase: annotation and discovery of microRNAs and other noncoding RNAs from deep-sequencing data. Methods Mol Biol. 2012; 822: 233–248. PubMed Abstract \| Publisher Full Text\n\nHuang PJ, Liu YC, Lee CC, et al.: DSAP: deep-sequencing small RNA analysis pipeline. Nucleic Acids Res. 2010; 38(Web Server issue): W385–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPantano L, Friedländer MR, Escaramís G, et al.: Specific small-RNA signatures in the amygdala at premotor and motor stages of Parkinson's disease revealed by deep sequencing analysis. Bioinformatics. 2016; 32(5): 673–681. PubMed Abstract \| Publisher Full Text\n\nPantano L, franpantano: lpantano/seqclusterviz: v0.1.1. 2019. http://www.doi.org/10.5281/zenodo.2556774\n\nTsuji J, Weng Z: DNApi: A De Novo Adapter Prediction Algorithm for Small RNA Sequencing Data. PLoS One. 2016; 11(10): e0164228. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "45803", "date": "18 Mar 2019", "name": "Xavier Bofill-De Ros", "expertise": [ "Reviewer Expertise MicroRNA biogenesis and isoforms" ], "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 by Pantano et al. describes a novel software, SeqclusterViz, to visualize and help the interpretation of small RNA-seq data mapped using Seqcluster. Moreover, the authors illustrate its usage with a case study of small RNAs dysregulated in the brain of patients with Parkinson’s disease. This software is of importance to help researchers do in depth analysis and compare the exact mapping of reads across multiple samples. The subject of this manuscript is interesting and seems adequate for indexing in F1000Research. The work looks solid although minor edits could be performed to improve the quality and usability of the SeqclusterViz.\nMinor comments:\nThe SeqclusterViz displays the sequence of the reads mapped to a precursor RNA such as a pri-miRNA. In particular, mature miRNA is well known for having isoforms (isomiRs) with the addition or internal edit to non-templated nucleotides. The field of isomiR study is of growing interest, SeqclusterViz will benefit from a display of non-templated nucleotides. A secondary structure prediction is implemented on SeqclusterViz, please describe in more the prediction method used. Include input parameters (such as if GU pairs at the end of helices) and outputs such as MFE, bracket-dot notation... Provide units for the Y and X axis in “Abundance profile along precursor”. Provide normalized read counts on “Table with Sequences”. Repair the path to make the button “Load example” active. The buttons of “Add filter” and “Change line” can’t be linked to the samples easily.\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": "4562", "date": "11 Apr 2019", "name": "Lorena Pantano Rubino", "role": "Author Response", "response": "Dear Xavier Bofill-De Ros,Thanks a lot to find time to review this article. I will take actions on all the points I can address in the next month and report an update as soon as possible.Cheers" } ] }, { "id": "45091", "date": "01 Apr 2019", "name": "Stefan Scholten", "expertise": [ "Reviewer Expertise Transcriptomics", "developmental biology", "reproduction" ], "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 reports on a visualization tool for seqcluster outputs, a software tool to characterise small transcriptome data. It is a smart tool for sRNA visualization. The interactive view makes it attractive, especially the visualization of secondary structures. The performance of the tool fits description and the filter option is very helpful for jumping to the desired information.\n\nThe tool is restricted to seqcluster.db file as input and cannot be used as a general-purpose sRNA visulization tool using map files.\nSufficient information is provided to allow interpretation of the expected output data sets and results generated with the tool. The example expression profile provides clear distinction between samples.\nThe editing option makes it even better, but when one switches to the line view different lengths can be seen. It is not clear whether this is related with the length of the sRNA that map at that position. A summary of the lengths that mapped would be useful additional information. Alongside the description section in Figure A (left side) information on the length of sRNA 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? 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": "4561", "date": "11 Apr 2019", "name": "Lorena Pantano Rubino", "role": "Author Response", "response": "Dear Stefan Scholten,Thanks for finding time to review this article. I will work on your recommendation to add that information to the tool, it is a very good idea we missed.I'll post back the update version here.Cheers" } ] } ]	1	https://f1000research.com/articles/8-232
https://f1000research.com/articles/8-972/v1	26 Jun 19	{ "type": "Opinion Article", "title": "Interstitial cystitis/bladder pain syndrome research: the answer may be just around the corner", "authors": [ "Vicki Ratner" ], "abstract": "Despite tremendous efforts, a large cadre of excellent researchers have been unable to definitively identify any cause of interstitial cystitis/bladder pain syndrome (IC/BPS) or develop more effective treatments. Newer research suggests that IC/BPS may have an infectious etiology. IC/BPS may also be related to mast cells, which have not been re-evaluated adequately. Many of the new investigative techniques, such as DNA/genomic analysis, microbiomes, and mycobiomes, applied from the fields of microbiology, infectious diseases, gastroenterology and mast cell specialists, have not have been fully utilized and applied to the field of urology and the study IC/BPS. Additional collaboration with these other fields of medicine may have a substantial impact on IC/BPS research and will likely move the urology community much closer to the causes of, and possible cures for, this most debilitating disease.", "keywords": [ "Interstitial Cystitis/Bladder Pain Syndrome IC/BPS", "Bladder Pain Syndrome (BPS)", "Interstitial Cystitis (IC)", "IC/BPS Research", "IC/BPS and biofilms", "IC/BPS and mast cells" ], "content": "Introduction\n\nThe first article on interstitial cystitis (IC) was published in the United States in 18871. However, research in earnest in the U.S. did not begin until the first National Institutes of Health (NIH) meeting held one hundred years later, in 1987. Although much progress has been made since that time, the cause and cure of interstitial cystitis/bladder pain syndrome (IC/BPS) remain elusive.\n\nIC affects up to 10 million people in the U.S2–4. Although it affects both sexes, it is more common in females2–4. Symptoms of urinary urgency, frequency and pain can vary from mild to severe and intermittent to constant. It is unclear if IC/BPS is one disease, one disease associated with other conditions, such as chronic fatigue syndrome, Crohn’s disease, irritable bowel syndrome, endometriosis and vulvodynia, for example5, or a systemic condition with a urinary or hematologic marker yet to be found. This paper aims to take another look at infectious agents and mast cells, both of which have been shown to be key components in other medical conditions, such as Crohn’s disease and mast cell activation syndrome (MCAS), which have not been adequately re-assessed in the field of urology.\n\n\nFuture directions in research\n\nThere are many similarities between the lining of the genitourinary tract and gastrointestinal tract. Pathologic extracellular organisms have been identified on the cells lining the gastrointestinal tract, but have yet to be been found on the urothelial lining of the bladder. A search for other pathologic extracellular organisms on IC/BPS urothelium using modern investigative techniques may prove enlightening. It may hold the key to identifying at least some etiologies of IC/BPS and could lead to treatments that may dramatically reduce symptoms or possibly lead to a cure.\n\nFamilial Crohn’s Disease is a small subset of Crohn’s Disease. Although admittedly a small sample size was used, in 2017, researchers reported the presence of three organisms (Candida tropicalis, Escherichia coli, and Serratia marcescens) on thelining of the gastrointestinal tract, detected using DNA sequencing6. Many organisms stick to the lining of the intestines via fimbriae and produce biofilms to protect themselves; thus, allowing these organisms to escape the body’s normal protective immune responses and form an impenetrable barrier to antibiotics. Recently (April 2019), it was shown that the above three organisms were found in elevated amounts in a cohort of patients with Crohn’s Disease and that the use of specific probiotics significantly decreased the number of these pathologic organisms; thereby reducing the amount of inflammation that they caused7.\n\nIn additional to taking a bladder biopsy, urologic researchers could further investigate IC/BPS patients whose urine cultures are negative, despite the patients being clinically symptomatic. PCR is one such technique. Another would be to take a second sample from these patients, spin down the urine samples, pour off the supernatant, and look at the remaining urothelial cells using a confocal scanning laser microscope to determine whether there are any bacteria or fungi attached to the cells (personal communication with Dr. Mamoud Ghannoum, Case Western Reserve University). If this technique reveals that specific organisms are present on a large number of IC/BPS urothelial cells, and not in controls, larger studies should be reproduced at other medical centers in order to validate this technique.If positive, this may indicate that IC/BPS has an infectious etiology, despite negative urine cultures.\n\nThe next step could be to create specific phages (lytic viruses) that could penetrate the biofilms of specific organisms that might be found on these urothelial cells, followed by administration of appropriate antibiotics. This might be one way to cure IC/BPS if extracellular organisms are the cause8,9.\n\nMast cells reside in all vascularized tissues of the body, including the bladder. They are generally found in close proximity to blood vessels and nerves. Mast cells contain over 200 types of granules/mediators. The hypothesis that inappropriate chronic mast cell activation may be an integral element, and perhaps even the root cause of IC/BPS in at least some patients, should be considered and revisited employing updated techniques10–13. The mast cell biopsy should be re-evaluated in the field of IC/BPS using the stain most specific for mast cells, CD117. In addition, an accurate measurement of the levels of these mast cell granules (e.g. tryptase, histamine, N-methylhistamine, heparin and prostaglandin D2, among others) many of which have quite short half-lives and great thermolability) in urine samples has been quite challenging to determine and should be re-assessed14–16.\n\nThis hinders or even renders impossible, the ability to demonstrate in the IC/BPS population that mast cell activation exists. Laboratory techniques have improved with time and assessment of IC/BPS patients for biochemical evidence of mast cell activation is becoming more feasible, especially using CD117 staining10–13. Although a paper published in 2015 addressed a great deal of research on mast cells and mast cell activation syndrome (MCAS), it was not specific for IC/BPS17.\n\nIf mast cell mediators can be consistently found in the urine of IC/BPS patients, then inappropriate mast cell content release might explain the etiology of IC/BPS patients, or at least one etiology of IC/BPS. This might provide additional means for diagnostic testing and/or be a marker for the condition. It may also point towards more effective treatment directed at specific inflammatory mast cell mediators, reduce time to diagnosis, and provide the basis for at least one component of a classification system for the disease.\n\nSimply relying on the number of mast cells seen on biopsy is not enough. The number of mast cells may not be as important as their level of activation and/or degranulation. Mast cells may be hyper-responsive and degranulate more frequently in response to variable trigger stimuli or may selectively release specific inflammatory mediators in response to a certain type of stimulus. The release of inflammatory mediators without degranulation of the mast cell may also occur10–13. Proper histochemical and/or immunohistochemical staining of biopsied tissue is critical for revealing the mast cells therein, as these cells usually cannot be recognized as mast cells with routine hematoxylin and eosin staining, instead being mistaken for other types of cells such as lymphocytes or macrophages. The best staining for mast cells is CD117 immunohistochemical staining, which is independent of the mast cell’s state of activation and is seen brightly on mast cells (a pattern seen on virtually no other cells). Certain other histochemical stains, such as tryptase and toluidine blue, have long been used by pathologists to identify mast cells. However, these stains principally target the mast cells’ granules; thus, they may not be as revealing for MCAS as CD117 can be, given that MCAS is a disease whose dominant feature is inappropriate mast cell activation and whose cellular-level pathology is dominated by mast cell degranulation, including complete degranulation which leaves an ‘empty’ cell that can be identified10–13.\n\nIn addition to degranulation, mast cell contents are able to transgranulate to nerves via the formation of filipodia (thin, finger-like projections) that attach directly to the neuronal membranes of nerves, including nerves within the bladder. The contents of the mast cells empty directly into the nerve cells via endocytosis. Transgranulation of mast cell contents to nerves in the brain was shown via time-lapse photography using an electron microscope in 200518. It is possible that sensory nerves in the bladder are triggered by mediators released from mast cells (either in the bladder, elsewhere or both), generating impulses that travel to the spinal cord and, from there, to the pain centers in the brain. This may be an explanation for the pelvic pain symptoms seen in many patients with IC/BPS.\n\n\nConclusion\n\nMoving forward, it is critical that researchers in urology think ‘outside the box’ and increase their collaboration with researchers in other fields of medicine that may relate to IC/BPS, such as gastroenterology, microbiology, infectious disease, and mast cell specialists. Possibilities discussed in detail in this paper include extracellular organisms that may form biofilms on the surface of the urothelium of the bladder lining (using Crohn’s Disease as a model). This can result in a negative urine culture, yet the painful bladder symptoms might still be caused by an infectious agent. Mast cells may play a much larger role in IC/BPS than previously thought. Hopefully, in the future, National Institutes of Diabetes, Digestive and Kidney (NIDDK) grants will make infection and mast cell involvement in IC/BPS a high priority in their research portfolio.\n\nIt is important to keep in mind how far we have to go, how much misery this condition is still causing, and how many hundreds of thousands of lives IC/BPS continues to ravage. The pain can be so intolerable that some patients have been driven to take their own lives19. Additional research on areas discussed in this paper should be undertaken and other areas re-evaluated. Since 1987, when the first NIDDK conference on IC was held, over 30 years have come and gone. The need for adequate treatments and a cure are urgent, yet little practical help for patients has been found20.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAckowledgements\n\nThe author would like to thank Lawrence Afrin, M.D. for his medical editorial assistance, Professor Gayle Greene for her review and editorial assistance, and Gerry Buena, Stanford Medical Library, for her medical research.\n\n\nReferences\n\nSkene AJC: Diseases of Bladder and Urethra in Women. New York: WM Wood; 1887; 167. Reference Source\n\nBerry SH, Elliott MN, Suttorp M, et al.: Prevalence of symptoms of bladder pain syndrome/interstitial cystitis among adult females in the United States. J Urol. 2011; 186(2): 540–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKonkle KS, Berry SH, Elliott MN, et al.: Comparison of an interstitial cystitis/bladder pain syndrome clinical cohort with symptomatic community women from the RAND Interstitial Cystitis Epidemiology study. J Urol. 2012; 187(2): 508–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClemmens JQ, Link CL, Eggers PW, et al.: Prevalence of painful bladder symptoms and effect on quality of life in black, Hispanic and white men and women. J Urol. 2007; 177(4): 1390–4. PubMed Abstract \| Publisher Full Text\n\nAlagiri M, Chottiner S, Ratner V, et al.: Interstitial cystitis: unexplained associations with other chronic disease and pain syndromes. Urology. Elsevier. 1997; 49(5A Suppl): 52–7. PubMed Abstract \| Publisher Full Text\n\nHoarau G, Mukherjee PK, Gower-Rousseau C, et al.: Bacteriome and Mycobiome Interactions Underscore Microbial Dysbiosis in Familial Crohn's Disease. mBio. 2016; 7(5): pii: e01250-16, 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHager CL, Isham N, Schrom KP, et al.: Effects of a Novel Probiotic Combination on Pathogenic Bacterial-Fungal Polymicrobial Biofilms. mBio. 2019; 10(2): Pii: e00338-19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKortright KE, Chan BK, Koff JL, et al.: Phage Therapy: A Renewed Approach to Combat Antibiotic-Resistant Bacteria. Cell Host Microbe. @ Elsevier, 2019; 25(2): 219–232. PubMed Abstract \| Publisher Full Text\n\nChan BK, Abdeon ST: Bacteriophages and their enzymes in biofilm control. Curr Pharm Des. 2015; 21(1): 85–99. PubMed Abstract \| Publisher Full Text\n\nRatner V: Mast cell activation syndrome. Transl Androl Urol. 2015; 4(5): 587–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfrin LB, Molderings GJ: A concise, practical guide to diagnostic assessment for mast cell activation disease. World J Hematol. 2014; 3(1): 1–17. Publisher Full Text\n\nMolderings F, Brettner S, Homann H, et al.: Mast cell activation disease: a concise practical guide for diagnostic workup and therapeutic options. J Hematol Oncol. 2011; 4: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfrin LB: A new era for an old cell: heightened appreciation of mast cell disease emerges. In: In-Depth Review: Mast Cell Disease: Beyond Allergy and Mastocytosis. Elsevier, 2016; 1–4. PubMed Abstract \| Publisher Full Text\n\nSant GR, Theoharides TC: The role of the mast cell in interstitial cystitis. Urol Clin North Am. 1994; 21(1): 41–53. PubMed Abstract\n\nel-Mansoury M, Boucher W, Sant GR, et al.: Increased urine histamine and methylhistamine in interstitial cystitis. J Urol. 1994; 152(2 Pt 1): 350–3. PubMed Abstract \| Publisher Full Text\n\nTheoharides TC, Sant GR, el-Mansoury M, et al.: Activation of bladder mast cells in interstitial cystitis: a light and electron microscopic study. J Urol. 1995; 153(3 Pt 1): 629–36. PubMed Abstract \| Publisher Full Text\n\nTheoharides TC, Valent P, Akin C: Mast Cells, Mastocytosis, and Related Disorders. N Engl J Med. 2015; 373(2): 163–72. PubMed Abstract \| Publisher Full Text\n\nWilhelm M, Silver R, Silverman AJ: Central nervous system neurons acquire mast cell products via transgranulation. Eur J Neurosci. 2005; 22(9): 2238–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRatner V: Placing Interstitial Cystitis/Bladder Pain Syndrome on the Map: The Story of the Interstitial Cystitis Association. In: Hanno PM, Nordling J, Staskin DR, WeinAJ, Wyndaele JJ, edts. Bladder Pain Syndrome. – An Evolution. London, Springer; 2018; chapter 35. : 155–160. Publisher Full Text\n\nHanno P, Wein A: Afterward. In: Hanno PM, Nordling J, Staskin DR, Wein AJ, Wyndaele JJ edts. Bladder Pain Syndrome – An Evolution. London, Spinger; 2018; Chapter 37. : 165–166. Publisher Full Text" }	[ { "id": "50452", "date": "08 Jul 2019", "name": "Christopher K. Payne", "expertise": [ "Reviewer Expertise I am fellowship trained in Female Urology and Pelvic Reconstructive Surgery. I was the Director of Female Urology at Stanford for over 20 years. I am now in a private practice focused on Urologic Chronic Pelvic Pain." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 hopeful article, pointing out the many exciting areas where scientific advances might be applied to better understand and treat patients with chronic bladder pain. Unfortunately, while the individual statements are factually correct, the guiding vision is rooted in the same narrow conception of disease that has produced so many failed studies over the past decades. The author acknowledges that IC/BPS is not well understood, \"It is unclear if IC/BPS is one disease, one disease associated with other conditions, such as chronic fatigue syndrome, Crohn’s disease, irritable bowel syndrome, endometriosis and vulvodynia, for example, or a systemic condition with a urinary or hematologic marker yet to be found.\"\n\nIn 1978, Meares and Stamey hypothesized that \"petechia-like hemorrhages (glomerulations) on the second distention of the bladder is the hallmark of interstitial cystitis, and that a reduced bladder capacity and a Hunner's ulcer represent a different (classic stage of the disease).1\" From this point glomerulations became the key to diagnosis (even worse, now we rely only on symptoms to diagnose BPS). However, this hypothesis was wrong. After over 40 years there is not even a single case report of a patient progressing from glomerulations to classic IC. A recent review concluded that there was, \"no convincing evidence . . . that glomerulation should be included in the diagnosis or phenotyping of BPS/IC.2\"\nIn fact, classic ulcerative Interstitial Cystitis (IC) is almost certainly a unique disease and, if thought leaders were to acknowledge it as such, great progress might be made in this area. The author's approaches might even pay off in this population.\n\nOn the other hand, Bladder Pain Syndrome (BPS) is no more than what its name implies--a syndrome, a group of patients with similar symptoms. This may be useful for epidemiological purposes, but it is of little value clinically. These patients have many different individual causes for their symptoms. It is usually possible to identify the primary symptom drivers in individual patients and, using this, treat them successfully. Individual patients can achieve remission with therapies as diverse as bladder instillations, myofascial physical therapy, and treatment aimed at central sensitization. It does not make sense to enrol such diverse BPS patients in clinical or research trials; in particular, there is no rationale to combine IC and BPS in any type of research protocol.\nI agree with the author that, \"researchers in urology think ‘outside the box’ and increase their collaboration with researchers in other fields of medicine\". However, studies should be done with reasonably homogeneous groups of patients and using appropriate hypotheses. It may well be worth revisiting mast cell abnormalities in cohorts of patients with multiple allergies. Advances in infectious disease might plausibly be applied to those classic IC or a clear bladder-centric phenotype. A great many patients have risk factors for and evidence of central sensitization. The NIDDK MAPP study group is exploring such patients and has already made many relevant observations. As the author emphasizes, \"It is important to keep in mind how far we have to go, how much misery this condition is still causing, and how many hundreds of thousands of lives IC/BPS continues to ravage.\" Nevertheless, research funding and effort is a scarce commodity and must be directed wisely. Continuing to use the same paradigm that has produced so little will not be wise.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [ { "c_id": "4804", "date": "12 Aug 2019", "name": "Vicki Ratner", "role": "Author Response", "response": "I was perplexed by Chris Payne's review of my opinion article. Most of the review addressed issues I did not raise. My editorial was not about the diagnosis of IC or the differences between IC and BPS. IC/BPS is the standard terminology used when discussing IC. While the reviewer's comments are important, they did not speak to the concerns raised by my article.Regarding the statement that the focus of suggestions in my paper were 'too narrow', I was suggesting that two areas receive additional focus: infection and mast cells. I did not mean to infer that my suggestions warranted a costly grant from NIH. Perhaps I did not make myself clear. The suggestions presented were meant to be small pilot studies to see if the results were worth pursuing." } ] }, { "id": "51310", "date": "24 Jul 2019", "name": "Sandor Lovasz", "expertise": [ "Reviewer Expertise Treatment of IC/BPS", "especially developing new way of local drug delivery and less expensive treatment modalities." ], "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 publication is not a report on the results of a clinical or a basic scientific study. Its an opinion article by the category. We do not have to expect proof of hypotheses, new findings, or innovation. However, we get the logically well-founded opinion of an author, who has been deeply involved in the topic of interstitial cystitis for more than three decades. She points out two challenging, yet not wholly cleared fields of the etiopathogenesis of IC/BPS: namely the role of mastocytosis and the possible presence of unidentified \"bacteria or fungi attached to the cells”.\nMuch research was performed worldwide, and many publications are available regarding these two suggested topics, but no conclusion could be drawn. This and the newly available sophisticated lab techniques make still sense to revive these research works.\n\nYet I think, there are a series of other (maybe even more compelling) research topics: to define the role of microbiome in IC/BPS, to develop more objective and non-invasive diagnostic tools, to find the most efficacious composition of the bladder cocktails, to reduce the costs and invasiveness of the IC/BPS therapy, to work out a unified long-term follow-up system of the patients’ condition, etc.\nI agree entirely with the author on the importance of future cooperation with other specialities of medicine like \"gastroenterology, microbiology, infectious disease, and mast cell specialists” as it is mentioned, but also with gynaecologists, pharmacologists, dietetics, physiotherapists, psychologists, etc. There is an evident need for fruitful interactive teamwork, yet it is doubtless for the majority of researchers dealing with IC/BPS, that a well-functioning, complete team is nowadays rather a dream than reality.\nEverybody knows that certain negative tendencies are setting back the optimal flow of the desired research work. Basic research is less attractive for the younger generation of urologists than laparoscopy, robotics, radical tumour-surgery, infectiology, etc.\nIn summary, I can state that the thoughts of the author are worth to be considered. She does not want to propose a complete program for IC/BPS research, but even in this limited extent, her opinion article is enormously essential to raise the awareness and to direct the attention of the society to this disease. Therefore, I definitely support this article to 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? 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/8-972
https://f1000research.com/articles/7-323/v1	15 Mar 18	{ "type": "Research Article", "title": "Assessment of the Cambridge Neuropsychological Test Automated Battery test in Saudi children with learning disabilities: A case-control study", "authors": [ "Nouf Al Backer", "Koloud Ateeq Alharbi", "Abdulrahman Alfahadi", "Syed Shahid Habib", "Shahid Bashir", "Nouf Al Backer", "Koloud Ateeq Alharbi", "Abdulrahman Alfahadi", "Syed Shahid Habib" ], "abstract": "Background: The neuropsychological tests and its subtests are composed of the motor planning task; simple reaction time task and the intradimensional/extradimensional shift (IED) task from the Cambridge Neuropsychological Test Automated Battery (CANTAB) were developed to examine specific components of cognition. The main objective of this study was to examine the reliability of these CANTAB subtests in pediatric patients with learning disabilities (LD) in Saudi Arabia. Methods: We administered the CANTAB subset test to 92 participants with LD and 68 controls with no LD. The tests performed were motor planning task (MOT), simple reaction time task (SRT) and the intradimensional/extradimensional shift (IED). Results: There was no significant age difference between the case and the control group (case: 9.2 ± 2.4 years versus controls: 9.0 ± 1.6 years, p=0.544). The IED and MOT were significantly longer among patients with LD versus control (p <0.001). LD cases had a longer SRT time than controls (cases: 1050.4 ± 626.5 versus controls: 815.5 ± 133.9, p=0.003). LD patients completed an average of 3.0 stages, than the controls, who were able to complete a mean of 8.4 IED stages (p<0.001). SRT was significantly longer in the case group (965.9 ± 716.4) compared to the controls (747.7 ± 120.7, p=0.014). LD cases made more errors in the motor screening tasks (MOT-Error) compared to the control group (case: 14.6 ± 4.5 versus controls: 12.4 ± 2.7, p<0.001). Conclusion: Patients with LD have poor CANTAB subtest results. If these CANTAB subtests do measure cognitive function, this adds to the accumulating evidence of cognitive impairment association in LD, and such studies should remain an active area of research.", "keywords": [ "CANTAB", "learning disabilities", "cognition", "Saudi Arabia" ], "content": "\n\n\n\n18th October 2019: Since publication of this article, the F1000Research editorial team were made aware of potential issues with the manuscript and the accompanying data that could affect interpretation of the results. We are investigating these issues with corresponding author Shahid Bashir, in line with guidance from the Committee of Publication Ethics. Further action will be dependent on the outcome of these discussions and peer review activity has been suspended in the meantime. We request that no one copies or redistributes the material in any medium or format until this issue is resolved.\n\n11th November 2019: Since being made aware of the potential problems with the data in this article, the F1000Research editorial team have continued to communicate with corresponding author, Shahid Bashir, in attempt to identify and promptly resolve the issues identified. After further investigation and discussion with co-authors, Dr Bashir has provided evidence to show that the current findings are inaccurate as a result of honest error in the handling and transfer of data files. Dr Bashir is working closely with the editorial team to correct and reanalyse the data from the raw files. In line with F1000Research policies for permanency of content, we will soon be publishing a revised ‘corrected’ version of this article to ensure the data are corrected and fully reliable. This policy takes into account current best practice in the scholarly publishing and library communities, and ensures the integrity and completeness of the scholarly record. Corrections and changes relative to the previous version will be clearly summarised in the ‘Amendments’ section at the start of the new version.\n\n\nIntroduction\n\nCognitive impairment of executive dysfunctions (EFs) is very common in individuals diagnosed with learning disabilities (LD)1–3. The EFs profile contains several brain functions including planning, organization, self-monitoring, mental representation of tasks essential driven to prefrontal cortex to perform complex behavior task throughout life2,4–6. There are a number of experimental studies showing EFs in individuals with LD disorders, across a wide range of functional levels and ages. These studies have reported that response inhibition, working memory, cognitive flexibility, planning, fluency and vigilance problems in these children7–10. EFs, including response inhibition, set-shifting, working memory, and planning, have been found to be impaired in children with attention deficit hyperactivity disorder (ADHD) and with LD10–13.\n\nThe computer-controlled system known as the Cambridge Neuropsychological Test Automated Battery (CANTAB) is a nonverbal (visually presented) set of tasks developed to recognize areas of the brain for cognition14–21.\n\nThree CANTAB subtests, the Motor task, simple reaction time, and the Intradimensional/Extradimensional (IED) tasks, test the functions of prefrontal cortex region of the brain, which contains the planning and programming areas of the human brain. Neuroimaging data has provided support in exploring their roles. Thus, cognitive impairments associated with LD, and their neural underpinnings can be studied by CANTAB.\n\nEFs refers to neuropsychological processes that enable physical, cognitive, and emotional self-control in LD children. EFs are often present in neurodevelopmental disorders, but examinations of the specificity of these deficits and direct comparisons with age matched controls are few. Therefore, we conducted the current study investigated EFs in children with LD in comparison to age matched healthy controls. We hypothesized that the LD group would have more severe cognitive dysfunctions than healthy controls.\n\n\nMethod\n\nRecruitment and testing of participants with learning disorders took place at the King Saud University Medical City (KSU-MC), Riyadh, Saudi Arabia. Participants were recruited for both case and controls, were aged between 6 and 15 years old from the neurology consultation clinics of KSU-MC coming for their follow up visits. The study was conducted from June 2016 to December 2017. A case (test) group was recruited together with a control group. The case group was formed of diagnosed patients with neurodevelopmental disorders, particularly those with learning disabilities and ADHD. Neurology consultants performed extensive interviewing for the control group, which was formed of individuals without neurodevelopmental conditions, psychopathology, or learning disabilities. Control group consisted of healthy children with age matching with LD group. These subjects did not have any family history of LD. They were recruited from pediatric clinics when visiting for follow up appointments.\n\nWritten consent was obtained from the parents of the patients upon joining the study. Ethical consent was obtained from the Institutional Review Board of the College of Medicine, King Saud University, Riyadh, Saudi Arabia [Project No: E-13-983].\n\n\nCANTAB testing\n\nThe three subtests from the CANTAB computerized battery were administered and responses were recorded directly with a touch-sensitive screen. Multiple training trials to learn the requirements of each task were given to each participant.\n\nBefore the actual test is started orientation trials were given to familiarize the subjects with the tests. The coinvestigators were trained for CANTAB testing and they administered the tests to the participants under the supervision of their consultant.\n\nMOT task provides an assessment (speed, accuracy and number of errors) involving the selection of colored crosses in different locations on the screen as quickly and accurately as possible by the participant.\n\nIED is a test that assesses the shifting and flexibility of attention in the fronto-striatal areas of the brain and takes about 7 minutes. There are two dimensions that are used in this test: color-filled shapes and white lines. The simple stimuli are the color-filled shapes and the compound stimuli are both the color-filled shapes and the white lines. This test started with two simple stimuli appearing in the screen and the subject has to learn the correct stimuli and respond by touching it. Feedbacks teach the subject the correct stimuli. After 6 correct responses, the stimuli and/or the rule changes. IED test assesses the errors, and the number of trial and stages completed. The detail description of the task described above is available from the CANTAB website.\n\nAnalysis of the data were performed by Statistical Package for Social Sciences (SPSS) version 23 (SPSS Inc., IBM, Chicago, Illinois, USA). The CANTAB sets and subsets between cases and controls were compared using an independent t-test. A p value of <0.05 was considered statistically significant.\n\n\nResults\n\nA total of 160 participants participated in the study, 92 (57.5%) for the case group and 68 (42.5%) in the control group. The participants in control group were less because age matched selection was difficult. The mean age for all participants was 9.1 ± 2.1 years, ranging from 6 to 15 years old. Table 1 shows the demographic characteristics and the results of the CANTAB test and subsets of all participants. There were no significant differences between the case and the control group (case: 9.2 ± 2.4 years versus controls: 9.0 ± 1.6 years, p=0.544)\n\nCambridge Neuropsychological Test Automated Battery (CANTAB), motor planning task (MOT), simple reaction time task (SRT), intradimensional/extradimensional shift (IED), Standard deviation (SD), Percent (Per) and Median (MED).\n\nTable 2 shows the comparison of the three CANTAB subsets between cases and controls. The IED and MOT were significantly greater/longer among patients with learning disabilities (cases) compared to those without a disability (control). P values were <0.001 and <0.001, respectively. Patients with learning disabilities (cases) had a longer SRT time than controls (cases: 1050.4 ± 626.5 versus controls: 815.5 ± 133.9, p=0.003).\n\nCambridge Neuropsychological Test Automated Battery (CANTAB), motor planning task (MOT), simple reaction time task (SRT), intradimensional/extradimensional shift (IED), Standard deviation (SD), Percent (Per) and Median (MED)\n\nTable 3 shows the results of further analysis of each of the CANTAB subsets between cases and controls. Those with learning disabilities (cases) were able to complete an average (mean) of 3.0 stages, which was significantly lower than the controls, who were able to complete a mean of 8.4 IED stages (p<0.001, Figure 1). The maximum time to react (SRT-Maximum) was significantly longer in the case group (965.9 ± 716.4) compared to the controls (747.7 ± 120.7), p=0.014, Figure 1. Patients with learning difficulties had significantly more errors in the motor screening tasks (MOT-Error) compared to the control group (case: 14.6 ± 4.5 versus controls: 12.4 ± 2.7, p<0.001, Figure 2). MOT-MED was significantly larger among the case group than the control group (p<0.001). SRT-Per was significantly lower among the cases than the control group (p<0.001, Figure 2).\n\nCambridge Neuropsychological Test Automated Battery (CANTAB), motor planning task (MOT), simple reaction time task (SRT), intradimensional/extradimensional shift (IED), Standard deviation (SD), Percent (Per) and Median (MED).\n\n\nDiscussion\n\nThe present study aimed to measure cognitive performance by using CANTAB tests for Saudi volunteers with and without a learning disability (LD). LD subjects had lower performances on tests of visual sustained attention, performance time, and learning abilities function in the present study. The key observations in our study were that children with LD have impairments in shifting and flexibility of attention (IED), general alertness, correct responses & errors of commission and omission (SRT) and motor performance (MOT).\n\nThese findings provide evidence that LD in early life is a risk factor for cognitive decline. Previous studies showed impairment in word fluency, memory and speech in LD populations9,10,22.\n\nTo our knowledge, this is the first study to study the cognitive function by CANTAB selected cognitive tests for Saudi LD volunteers. The IED tests, which activates the frontal cortex16,17,21 according to what we have mentioned earlier in IED explanation, and assesses working memory and strategy based on an individual's ability to retain, manipulate, and remember spatial information. The MOT test motivates the fronto-striatal circuits for sustained attention, while the SRT test activate the fronto-parietal functions and subcortical areas that regulate the planning, and execution of the motor action and psychomotor speed processing23,24.\n\nEducation levels is one factor of limitation in this study, which has been mentioned in previous study using of CANTAB system for cognition24 since they were admitted in different educational schools for LD children.\n\nIn the literature CANTAB and neuroimaging showed that reduced episodic memory s associated with diminished hippocampal volume, with reciprocal influences among neuroanatomical and cognitive variables25. The second limitation is that we did not use neuroimaging to correlate neuroanatomical changes with CANTAB results. The present evidence provides support of cognitive impairment association in LD and such studies should remain an active area of research.\n\n\nData availability\n\nDataset 1: Data containing test scores for all participants 10.5256/f1000research.13695.d19637926", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grant from Deanship of Scientific Research (RGP-1438-048) of King Saud University, Riyadh.\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\nAnderson PJ: Assessment and development of executive function (EF) during childhood. Child Neuropsychol. 2002; 8(2): 71–82. PubMed Abstract \| Publisher Full Text\n\nHughes C, Graham A: Executive functions and development. In: Reed J, Warner-Rogers J, editors. Child neuropsychology: Concepts, theory, and practice. Chichester, United Kingdom: Wiley-Blackwell; 2008; 264–84.\n\nDenckla MB: Measurement of executive function. In Frames of reference for the assessment of learning disabilities: New views on measurement issues. (ed. Reid Lyon G.) 1994; 117–142. (Paul H Brookes Publishing). Reference Source\n\nCasey BJ, Giedd JN, Thomas KM: Structural and functional brain development and its relation to cognitive development. Biol Psychol. 2000; 54(1–3): 241–57. PubMed Abstract \| Publisher Full Text\n\nAron AR: Progress in executive-function research: From tasks to functions to regions to networks. Curr Dir Psychol Sci. 2008; 17(2): 124–9. Publisher Full Text\n\nClark CA, Pritchard VE, Woodward LJ: Preschool executive functioning abilities predict early mathematics achievement. Dev Psychol. 2010; 46(5): 1176–91. PubMed Abstract \| Publisher Full Text\n\nGathercole SE, Alloway TP, Willis C, et al.: Working memory in children with reading disabilities. J Exp Child Psychol. 2006; 93(3): 265–81. PubMed Abstract \| Publisher Full Text\n\nHunter SJ, Sparrow EP: Executive Function and Dysfunction: Identification, Assessment and Treatment. (Cambridge University Press), 2012. Reference Source\n\nOzonoff S, Jensen J: Brief report: specific executive function profiles in three neurodevelopmental disorders. J Autism Dev Disord. 1999; 29(2): 171–177, (Date of access: 11/24/2015). PubMed Abstract \| Publisher Full Text\n\nCorbett BA, Constantine LJ, Hendren R, et al.: Examining executive functioning in children with autism spectrum disorder, attention deficit hyperactivity disorder and typical development. Psychiatry Res. 2009; 166(2–3): 210–222, (Date of access: 11/24/2015). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoldberg MC, Mostofsky SH, Cutting LE, et al.: Subtle executive impairment in children with autism and children with ADHD. J Autism Dev Disord. 2005; 35(3): 279–93. PubMed Abstract \| Publisher Full Text\n\nFuggetta GP: Impairment of executive functions in boys with attention deficit/hyperactivity disorder. Child Neuropsychol. 2006; 12(1): 1–21. PubMed Abstract \| Publisher Full Text\n\nSergeant JA, Geurts H, Oosterlaan J: How specific is a deficit of executive functioning for attention-deficit/hyperactivity disorder? Behav Brain Res. 2002; 130(1–2): 3–28, (Date of access: 02/16/2016). PubMed Abstract \| Publisher Full Text\n\nLuciana M: Practitioner review: computerized assessment of neuropsychological function in children: clinical and research applications of the Cambridge Neuropsychological Testing Automated Battery (CANTAB). J Child Psychol Psychiatry. 2003; 44(5): 649–663. PubMed Abstract \| Publisher Full Text\n\nOpen Science Collaboration: Psychology. Estimating the reproducibility of psychological science. Science. 2015; 349(6251): aac4716. PubMed Abstract \| Publisher Full Text\n\nSahakian BJ, Owen AM: Computerized assessment in neuropsychiatry using CANTAB: discussion paper. J R Soc Med. 1992; 85(7): 399–402. PubMed Abstract \| Free Full Text\n\nde Rover M, Pironti VA, McCabe JA, et al.: Hippocampal dysfunction in patients with mild cognitive impairment: a functional neuroimaging study of a visuospatial paired associates learning task. Neuropsychologia. 2011; 49(7): 2060–2070. PubMed Abstract \| Publisher Full Text\n\nRobbins TW, James M, Owen AM, et al.: A study of performance on tests from the CANTAB battery sensitive to frontal lobe dysfunction in a large sample of normal volunteers: implications for theories of executive functioning and cognitive aging. Cambridge Neuropsychological Test Automated Battery. J Int Neuropsychol Soc. 1998; 4(5): 474–490. PubMed Abstract \| Publisher Full Text\n\nDe Luca CR, Wood SJ, Anderson V, et al.: Normative data from the CANTAB. I: development of executive function over the lifespan. J Clin Exp Neuropsychol. 2003; 25(2): 242–254. PubMed Abstract \| Publisher Full Text\n\nBrett ZH, Humphreys KL, Fleming AS, et al.: Using cross-species comparisons and a neurobiological framework to understand early social deprivation effects on behavioral development. Dev Psychopathol. 2015; 27(2): 347–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith PJ, Need AC, Cirulli ET, et al.: A comparison of the Cambridge Automated Neuropsychological Test Battery (CANTAB) with \"traditional\" neuropsychological testing instruments. J Clin Exp Neuropsychol. 2013; 35(3): 319–328. PubMed Abstract \| Publisher Full Text\n\nSergeant JA, Geurts H, Oosterlaan J: How specific is a deficit of executive functioning for attention-deficit/hyperactivity disorder? Behav Brain Res. 2002; 130(1–2): 3–28, (Date of access: 02/16/2016). PubMed Abstract \| Publisher Full Text\n\nPersson J, Kalpouzos G, Nilsson LG, et al.: Preserved hippocampus activation in normal aging as revealed by fMRI. Hippocampus. 2011; 21(7): 753–766. PubMed Abstract \| Publisher Full Text\n\nLenehan ME, Summers MJ, Saunders NL, et al.: Does the Cambridge Automated Neuropsychological Test Battery (CANTAB) Distinguish Between Cognitive Domains in Healthy Older Adults? Assessment. 2016; 23(2): 163–172. PubMed Abstract \| Publisher Full Text\n\nPersson N, Ghisletta P, Dahle CL, et al.: Regional brain shrinkage and change in cognitive performance over two years: The bidirectional influences of the brain and cognitive reserve factors. Neuroimage. 2016; 126: 15–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl Backer N, Ateeq Alharbi K, Alfahadi A, et al.: Dataset 1 in: Assessment of the Cambridge Neuropsychological Test Automated Battery test in Saudi children with learning disabilities: A case-control study. F1000Research. 2018. Data Source" }	[ { "id": "41326", "date": "14 Dec 2018", "name": "Maksym V. Kopanitsa", "expertise": [ "Reviewer Expertise cognitive behaviour in preclinical disease models", "in vitro electrophysiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reviewed manuscript “Assessment of the Cambridge Neuropsychological Test Automated Battery test in Saudi children with learning disabilities: A case-control study” presents a useful dataset that compares performances of children with learning disabilities and those without them in several CANTAB tasks. These data confirm the notion that CANTAB can sensitively detect differences in executive function parameters in children with different learning ability. To enhance the value of the obtained data, I’d like to suggest that the authors consider the following points (perhaps revising their submission):\nIt is not quite clear what methods were used to diagnose children with LD.\n\nThe authors correctly cite the lack of neuroimaging data as a limitation of the study, however, perhaps they should mention if any of the subjects with LD had any genetic testing? Ideally, some statement about whether any of the subjects has syndromic or non-syndromic intellectual disability and results of any genetic tests should be included.\n\nWe believe that more studies where CANTAB was used for assessment of children with LD/ADHD could be discussed in the Discussion section. The Bibliography section on Cambridge Cognition web-site could be helpful in this regard: http://www.cambridgecognition.com/login/bibliography.\n\nMinor points:\nThe article should undergo careful language editing from the point of view of grammar and syntax. There are multiple instances of grammatically and contextually incorrect sentences. Below are very few selected examples:\na) The title of the study should be revised, because it was not an “Assessment of the …test in Saudi children”, but rather “Assessment of performance of Saudi children with learning disabilities by using the Cambridge Neuropsychological Test Automated Battery”.\nb) The second sentence of the Results section, “The participants in control group were less because age matched selection was difficult”, should be written as “There were fewer participants in control group because age matched selection was difficult”.\nc) The fifth sentence of the Results section should read “There were no significant differences in the ages between case and control groups (case: 9.2 ± 2.4 years versus controls: 9.0 ± 1.6 years, P = 0.544)” (“in the ages” was not mentioned in the original sentence).\nd) In addition, the term “EF” refers to “executive function” (which is correct) in some parts of the text and to “executive dysfunction” in other parts. For example: “EFs refers to neuropsychological processes that enable physical, cognitive, and emotional self-control in LD children” (function) but “There are a number of experimental studies showing EFs in individuals with LD Disorders…” (dysfunction).\n\nThere is no description of SRT in the methods.\n\nThe use of the Student’s t-test is contingent on the normal distribution of data in the compared groups, however there was no evidence this was checked prior to the comparison in this study.\n\nAs the data are clearly presented in the Tables, there is probably no reason to repeat the same numbers in the text. P-values also can be in a separate column in the Table. Likewise, because the data are quite straightforward, the Figures are not necessary as they just illustrate what is presented in the Tables. Ideally, we would recommend presenting the data as box and whisker plots with all individual values being represented by small dots. This would be the most informative format compared to current Tables and slightly less detailed Figures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4450", "date": "01 Mar 2019", "name": "Shahid Bashir", "role": "Author Response", "response": "The reviewed manuscript “Assessment of the Cambridge Neuropsychological Test Automated Battery test in Saudi children with learning disabilities: A case-control study” presents a useful dataset that compares performances of children with learning disabilities and those without them in several CANTAB tasks. These data confirm the notion that CANTAB can sensitively detect differences in executive function parameters in children with different learning ability. To enhance the value of the obtained data, I’d like to suggest that the authors consider the following points (perhaps revising their submission):1. It is not quite clear what methods were used to diagnose children with LD.We have added the statement “The case group was formed of diagnosed patients with neurodevelopmental disorders based on their medical records and following up with their regular child neurology consultant, particularly those with learning disabilities and ADHD”2. The authors correctly cite the lack of neuroimaging data as a limitation of the study, however, perhaps they should mention if any of the subjects with LD had any genetic testing? Ideally, some statement about whether any of the subjects has syndromic or non-syndromic intellectual disability and results of any genetic tests should be included.Thanks for this very valid point, but sorry we don’t have genetic assessment. We do genetic testing when we find parents or other siblings showing similar patterns. Secondly, as this was not part of the objectives of the study, we need to have ethical approval for getting genetic results.3. We believe that more studies where CANTAB was used for assessment of children with LD/ADHD could be discussed in the Discussion section. The Bibliography section on Cambridge Cognition web-site could be helpful in this regard: http://www.cambridgecognition.com/login/bibliography. We have revised the discussion part.Minor points:1. The article should undergo careful language editing from the point of view of grammar and syntax. There are multiple instances of grammatically and contextually incorrect sentences. Below are very few selected examples:a) The title of the study should be revised, because it was not an “Assessment of the …test in Saudi children”, but rather “Assessment of performance of Saudi children with learning disabilities by using the Cambridge Neuropsychological Test Automated Battery”.Thanks for the feedback. We have revised the title.b) The second sentence of the Results section, “The participants in control group were less because age matched selection was difficult”, should be written as “There were fewer participants in control group because age matched selection was difficult”.Done.c) The fifth sentence of the Results section should read “There were no significant differences in the ages between case and control groups (case: 9.2 ± 2.4 years versus controls: 9.0 ± 1.6 years, P = 0.544)” (“in the ages” was not mentioned in the original sentence).Done.d) In addition, the term “EF” refers to “executive function” (which is correct) in some parts of the text and to “executive dysfunction” in other parts. For example: “EFs refers to neuropsychological processes that enable physical, cognitive, and emotional self-control in LD children” (function) but “There are a number of experimental studies showing EFs in individuals with LD Disorders…” (dysfunction).Done.2. There is no description of SRT in the methods.Thanks for your feedback and we really apologise for this mistake. We have added this in methods.3. The use of the Student’s t-test is contingent on the normal distribution of data in the compared groups, however there was no evidence this was checked prior to the comparison in this study.4. As the data are clearly presented in the Tables, there is probably no reason to repeat the same numbers in the text. P-values also can be in a separate column in the Table. Likewise, because the data are quite straightforward, the Figures are not necessary as they just illustrate what is presented in the Tables. Ideally, we would recommend presenting the data as box and whisker plots with all individual values being represented by small dots. This would be the most informative format compared to current Tables and slightly less detailed Figures.We have removed the table and figures too." }, { "c_id": "4675", "date": "29 May 2019", "name": "Shahid Bashir", "role": "Author Response", "response": "TITLE: Assessment of performance of Saudi children with learning disabilities by using the Cambridge Neuropsychological Test Automated Battery. Introduction: The recent meta-analysis have shown that the CANTAB tasks have a well-established sensitivity to a wide range of cognitive effects including ADHD 22. Method The case group was formed of diagnosed patients with neurodevelopmental disorders based on their medical records and following up with their regular child neurology consultant, particularly those with learning disabilities and ADHD. SRT The Simple Reaction Time task measures simple reaction time, general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The only uncertainty is regarding when the stimulus will occur, by having a variable interval between the trial response and the onset of the stimulus for the next trial. Outcome measures cover latency (response speed), correct responses and errors of commission and omission. Results: There were fewer participants in control group because age matched selection was difficult. The IED and MOT were significantly greater/longer among patients with learning disabilities (cases) compared to those without a disability (control) (Table 1). Patients with learning disabilities (cases) had a longer SRT time than controls (p=0.003). Table 2 shows the results of further analysis of each of the CANTAB subsets between cases and controls. Those with learning disabilities (cases) were able to complete an average (mean) of 3.0 stages, which was significantly lower than the controls, who were able to complete a mean of 8.4 IED stages (p<0.001, Table 2). The maximum time to react (SRT-Maximum) was significantly longer in the case group (965.9 ± 716.4) compared to the controls (747.7 ± 120.7), p=0.014, Table 2. Patients with learning difficulties had significantly more errors in the motor screening tasks (MOT-Error) compared to the control group (case: 14.6 ± 4.5 versus controls: 12.4 ± 2.7, p<0.001, Table 2). MOT-MED was significantly larger among the case group than the control group (p<0.001). SRT-Per was significantly lower among the cases than the control group (p<0.001, Table 2). Discussion: A number of key limitations should be considered when interpreting the findings presented here. First, although the three CANTAB tasks used in this study were selected to evaluate potential cognitive effects in children with LD, complex and diversity of LD may have affected cognitive function in domains that were not tested. Second, because data were analysed at the group level only, potentially clinically significant effect of cognitive function were not examined at individual level. Third, in the literature CANTAB and neuroimaging showed that reduced episodic memory s associated with diminished hippocampal volume, with reciprocal influences among neuroanatomical and cognitive variables 26. Forth limitation is that we did not use neuroimaging to correlate neuroanatomical changes with CANTAB results. The present evidence provides support of cognitive impairment association in LD and such studies should remain an active area of research. As compared to traditional neuropsychological tasks, the computerized based CANTAB tasks have proven congruence with an accepted and validated measure of cognitive function in subject with EF impairment27. The CANTAB includes several different tasks with sensitivity to particular types of neurocognitive dysfunction. Furthermore, because the tasks are computerized, the CANTAB benefits from reliability of administration and practice effects are minimized by the use of parallel versions across all testing sessions. Conclusion: This pilot study provides platform for further investigation of cognitive impairment in children and adolescents with LD, and suggests that potential long-term cognitive function assessment may be worthy of further investigation in well-controlled studies." } ] } ]	1	https://f1000research.com/articles/7-323
https://f1000research.com/articles/8-964/v1	25 Jun 19	{ "type": "Research Article", "title": "Dermatoglyphical impressions are different between children and adolescents with normal weight, overweight and obesity: a cross-sectional study", "authors": [ "Adriano Alberti", "Emil Kupek", "Clarissa Martinelli Comim", "Carina Rossoni", "Myrna Alicia Ruiz Reyes", "Josiane Aparecida De Jesus", "Leoberto Ricardo Grigollo", "Bruna Becker da Silva", "Ubirajara Duarte dos Santos", "Renan Souza", "Gracielle Fin", "Elisabeth Baretta", "Rudy José Nodari Júnior", "Emil Kupek", "Clarissa Martinelli Comim", "Carina Rossoni", "Myrna Alicia Ruiz Reyes", "Josiane Aparecida De Jesus", "Leoberto Ricardo Grigollo", "Bruna Becker da Silva", "Ubirajara Duarte dos Santos", "Renan Souza", "Gracielle Fin", "Elisabeth Baretta", "Rudy José Nodari Júnior" ], "abstract": "Background: Obesity is a health condition that causes a great impact on public health. The aim of this study was to determine the association between dermatoglyphic characteristics and excessive weight in children and adolescents aged 10 to 19 years in the center-west region of Santa Catarina, Brazil. Methods: The sample comprised of 2,172 children and adolescents aged 10 to 19 years old of both sexes and from public and private teaching networks. Results: The results suggested a predictive marker of obesity, with a greater number of lines in left hand finger two (Mesql2) and a higher frequency of the whorl pattern in participants of a healthy weight, while the overweight group had a higher frequency of the radial loop pattern and the obese group had a higher frequency of the ulnar loop pattern. Conclusion: It was concluded that there may be different dermatoglyphic characteristics depending on the nutritional status of children and adolescents.", "keywords": [ "Dermatoglyphic", "Obesity", "Child", "Adolescent" ], "content": "Introduction\n\nObesity is a matter of constant concern for global health and is considered to be the second most common cause of preventable death (WHO, 2014). This condition has a multifactorial origin involving genetic and environmental factors (Martínez García et al., 2017) and is considered one of the major problems for public health around the world. By 2014, more than 1.9 million adults were overweight. Of these, 600 million were obese. From 1980 to 2013, the number of obese and overweight people increased by 27.5% among adults and 47.1% among children, which generated even more concern (Ng et al., 2014).\n\nThe predictive value of dermatoglyphic features (DGFs) in relation to a variety of diseases has been investigated for more than five decades, from the seminal work of Cummins and Midlo in 1961 (Bhat et al., 2012; Sharma & Sharma, 2012; Shetty et al., 2016). Dermatoglyphics is the scientific study of epidermal crescent patterns and several researchers from different fields, such as biology, anthropology, genetics, and medicine, are engaged in unraveling several unknown aspects of this field (Raniwala et al., 2017). In addition, dermatoglyphics has been used as a noninvasive diagnostic tool to detect or predict different medical conditions that have a fetal origin (Wijerathne et al., 2016).\n\nThe reason for the association between DGFs and health is the influence of epigenetics, which affects both (Yohannes, 2015). The formation of the former begins in the first trimester of pregnancy, during the 6th week, is completed after the 24th week of gestation and is formed according to the development and maturation of the central nervous system (Babler, 1991; King et al., 2009).\n\nAlthough DGFs have no causal relationship with health (Mittal et al., 2012), they may be used as a marker of health problems when there are associations that are consistent with the diseases of interest, this condition being essential for effective screening (Gupta & Karjodkar, 2013). In the case of obesity, this is a multifactorial (polygenic and environmental) condition where epigenetic factors of obesity can influence DGF patterns; therefore, these can be used as markers of obesity throughout life (Bhardwaj et al., 2015).\n\nOur aims were to determine the association between DGF characteristics and obesity in children and adolescents aged 10 to 19 years in the center-west region of the state of Santa Catarina, in the south of Brazil and investigate whether a dermatoglyphic marker can of obesity exists.\n\n\nMethods\n\nA cross-sectional study of children and adolescents aged 10 to 19 years, female and male, from public and private schools in the municipality of Joaçaba, Santa Catarina, Brazil. This study was submitted to the Ethics in Research (CEP) with Human Beings from Unoesc/Hust and was approved under protocol number 449.924.\n\nThe data belong to the laboratory evaluation database and exercise physiology measurements of the University of West Santa Catarina (Unoesc) of Joaçaba. This data storage bank has data from 3,074 individuals investigated in the school census in the years 2013 – 2014, performed by the Institute of Educational Studies and Research “Anísio Teixeira” with the purpose of monitoring the development of these children and adolescents. The inclusion criteria for this study were students aged between 10 and 19 years enrolled in a public or private network in primary or secondary education in the municipality of Joaçaba, Santa Catarina, who participated in school censuses conducted in the years 2013 – 2014. All individuals with incomplete data for variables such as weight and height, with anomalous fingerprints due to, for example, sweat or excessive dirt on the fingers, were excluded from the sample. The final sample consisted of 2,172 students, of which 1,166 were female and 1,006 were male.\n\nAccording to the National Institute of Educational Studies and Research Anísio Teixeira (INEP, 2013), of the students enrolled in primary and secondary education in 2013 in the municipality of Joaçaba, 3,193 students were enrolled in public schools and 1,733 in private schools. In 2014, 2,842 students were enrolled in the public school system and 1,839 in the private system. Based on the number of students enrolled in their respective years, the database represents 64% of the total number of students.\n\nAlthough the students were familiar with the tests performed, the protocols of each were detailed verbally by the evaluators in order to reduce the margin of error, with the exception of the dermatoglyphic test, which is only part of this study but is easy to perform.\n\nThe body mass index (BMI) tables of the Ministry of Health of Brazil (Ministry of Health, 2011) were used to classify BMI, dividing their percentages by age and sex, thus denominating them: low weight (< 5th percentile), healthy weight (≥ 5th percentile and < 85th percentile), overweight (≥ 85th percentile and < 97th percentile) and obese (≥ 97th percentile), according to the World Health Organization (WHO, 2014).\n\nThe anthropometric evaluation of children and adolescents consisted of three phases and was carried out in the following way: first, weight was measured by a single measurement in a calibrated digital scale, with a maximum capacity of 150 kilos (kg). The scale was supported on a flat, firm and smooth surface. The participant was positioned in the center of the scale, wearing the least possible clothing, barefoot, erect, feet together, arms extended along the body and looking at the horizon (Ministry of Health, 2011). Once their balance was stable, weight was recorded in kg.\n\nAfter weight was recorded, stature was measured using a vertical mobile stem stadiometer, with a scale in centimeters (cm) and an accuracy of one millimeter (mm). The patients were positioned with their backs to the instrument, barefoot, feet together, in an upright position, looking forward, with their arms extended along the body. The mobile part of the stadiometer was placed on the top of the head at the highest point and the height reading was performed (Ministry of Health, 2011).\n\nThe BMI was calculated using the following formula that relates weight (kg) to height (meters): BMI = Weight / Height(WHO, 1995).\n\nThe collection of the fingerprints occurred after the collection of the other information within the schools and was collected by the researchers. The protocol proposed by Cummins and Midlo in 1961 was chosen to analyze DGF characteristics. For the capture, processing and analysis of fingerprints, a computerized process for dermatoglyphic reading was used. The Dermatoglyphic Reader consists of an optical scanner that collects and interprets the image and constructs, in binary code, a dermatoglyphic drawing, which is processed by the reader’s specific software for the treatment and reconstruction of real and binarized images in black and white, as validated by Nodari Júnior et al. in 2008.\n\nAfter all the images have been collected, the reader user selects them one by one and defines specific points (nucleus and deltas), tracing the Galton Line, and the software, through specific algorithms, marks the intersections of the line with the digital lines. In this way, the reader provides the number of lines on each finger, as well as the type of fingerprint pattern. The software carries out this qualitative pattern identification and quantitative determination of lines, generating a Microsoft Excel worksheet containing the processed data (Nodari-junior et al., 2008).\n\nThis fingerprint analysis could also have been carried out using non-proprietary methods, such as the traditional method proposed by Cummins and Midlo using ink and paper.\n\nStatistical analyzes were performed using the STATA version 12.0. Analysis of variance (ANOVA) for DGF tested the null hypothesis that there was no difference in the number of finger lines between the weight groups. The differences were considered statistically significant at p <0.05. The chi-square test was used to test whether there was a difference between weight groups in the following variables: arch, radial loop, ulnar loop and whorl fingerprint patterns (Figure 1). The differences were considered statistically significant at p <0.05.\n\nNodari Junior and Fin authorized the reproduction of this image (taken from Nodari-Júnior & Fin, 2016), both are authors of this article.\n\n\nResults\n\nThe final sample for this study consisted of 2,172 students, of which 1,166 were female and 1,006 were male (Alberti, 2019). Students were aged between 10 and 19 years and enrolled in a public or private primary or secondary education in the municipality of Joaçaba, Santa Catarina-Brazil.\n\nAs for the quantitative fingerprint variables (the number of lines of each finger, the total number of lines on the fingers of the right hand, the total number of lines on the fingers of the left hand and the total number of lines on the fingers of both hands), it was observed that, on average, individuals who were of a healthy weight had a greater number of lines in relation to overweight and obese individuals in MET2 (left-hand finger two). (Table 1).\n\nNote: oneway ANOVA.\n\nOW, overweight; Mdsql, number of lines on right hand fingers; Mesql, number of lines on left hand fingers; Sqtle: sum of the number of lines on the left hand; Sqtld, sum of the number of lines on the right hand; Sqtl, sum of the number of lines on both hands.\n\nThe results obtained may suggest the presence of predictive markers for BMI in the researched population. For left-hand finger two (MET2), participants of a healthy weight presented with a higher frequency the whorl pattern, while overweight and obese participants had a higher frequency of the ulnar loop pattern; for left-hand finger three (MET3), participants of a healthy weight presented a higher frequency of the radial loop pattern, while overweight and obese participants had a higher frequency of the ulnar loop pattern; for left-hand finger four (MET4), participants of a healthy weight presented a higher frequency of the whorl pattern, while the overweight group presented a higher frequency of the radial loop and the ulnar loop pattern; for left-hand finger five (MET5), participants of a healthy weight presented a higher frequency of the arch pattern, while the overweight group presented a higher frequency of the radial loop pattern and the obese group had a higher frequency of the ulnar loop pattern; for finger one of the right hand (MDT1), participants of a healthy weight presented a higher frequency of the arch pattern, the overweight group had a higher frequency of the radial loop pattern, and the obese group had a higher frequency of the ulnar loop pattern; for right-hand finger three (MDT3), participants of a healthy weight had a higher frequency of the whorl pattern, while overweight and obese groups had a higher frequency of the ulnar loop pattern; for right-hand finger four (MDT4), participants of a healthy weight had the highest frequency of the whorl pattern, while the overweight group presented a higher frequency of the radial loop pattern and the obese group presented a higher frequency of the figure ulnar loop; for right-hand finger five (MDT5), participants of a healthy weight presented a high frequency of the whorl pattern, while the overweight group presented a high frequency of the radial loop pattern and the obese group presented a high frequency of the ulnar loop pattern. (Table 2).\n\nNote: chi-square test. OW, overweight; MET, left hand; MDT, right hand.\n\n\nDiscussion\n\nThis study suggests that a higher number of the total number of lines in Mesql2 may be a predictive marker of obesity and that a higher frequency of the whorl pattern may be found in people of a healthy weight, a higher frequency of the radial loop pattern may be found in overweight people and a higher frequency of the ulnar loop pattern may be found in obese people.\n\nAn increase in weight in children and adolescents can occur rapidly, causing, for example, low levels of cardiorespiratory and musculoskeletal aptitude and impairing the quality of life of these individuals (García-Hermoso et al., 2018). Several studies have begun to recognize epigenetic factors in obesity and, despite a relatively high heritability of non-syndromic common obesity (40–70%), the search for genetic variants that contribute to susceptibility has been a challenging task. Genome-wide association studies have dramatically changed the pace of detection of common variants involved in genetic susceptibility. By the year 2011, more than 40 genetic variants were associated with obesity. However, since these variants do not fully explain the heritability of obesity, other forms of variation, such as epigenetic markers, should be considered (Herrera et al., 2011).\n\nEvery organism is unique and has epigenetic traits that are inherited and generated in the womb. Studies have been conducted that are aimed at highlighting the influence of the gestation period and fetal environment for the development of diseases and conditions over a lifetime, such as obesity (Martínez García et al., 2017). The fetal development phase begins at the 9th week of gestation and goes through to the baby’s birth, the human gestation lasting on average 38 weeks (Dipietro, 2008). There are studies that reinforce that epigenetic influences have a strong association with the development of obesity (Ornellas et al., 2017).\n\nDermatoglyphics has its fundamental basis in this premise, being an epigenetic marker related to the period of fetal development (Yohannes, 2015). In addition to the fact that the fingerprints are intrinsically related to the central nervous system and can therefore reflect motor capacities inherited genetically and epigenetically for conditions that may have a marker expressed during this period of fetal development, fingerprint evaluation is a simple and practical method (King et al., 2009).\n\nIn one sample of 370 obese children, in a study to identify dermatoglyphic patterns in obese individuals and to discover the association between standard dermatoglyphics and obesity, a high frequency of the arch pattern was observed in the right thumb (Bhardwaj et al., 2015).\n\nIn another study, the authors (Oladipo et al., 2010) sought to determine the dermatoglyphic characteristics of obese Nigerian patients by comparing a group of 50 obese individuals (25 men and 25 women) with a group of 50 normal weight subjects (25 men and 25 women). The arch pattern was observed in the first digits of the right hand in 54.5% of obese men and 42.33% of obese women, whereas individuals with normal weight presented the figure more frequently.\n\nIn the city of São Paulo, Brazil, a survey (Pasetti et al., 2012) with 30 obese Brazilian women with a mean age of 46.1 ± 07.87 years, all with a BMI equal to or greater than 30, observed that participants presented a high frequency of the arch pattern, low frequencies of the ulnar loop pattern and a high frequency of the whorl pattern. These results corroborate the findings of several other authors (Bhardwaj et al., 2015; Oladipo et al., 2010) who also presented a predominance of the arch pattern in the obese group.\n\nThe present study utilized a sample of 2,172 individuals and the computerized method developed by Nodari Júnior, Heberle, Ferreira-Emygdio and Irany-Knackfuss in 2008, providing greater precision in the dermatoglyphic analysis. This method allows optimization of the analysis and greater reliability in the counting and marking of lines and designs. It allowed differentiation of the ulnar loop and radial loop patterns, which other studies in dermatoglyphics and obesity have not done.\n\nThe results showed the presence of different dermatoglyphic characteristics for different nutritional statuses of children and adolescents, indicating a higher number of the total number of lines in Mesql2 and a higher frequency of the whorl pattern may be found in people of a healthy weight, a higher frequency of the radial loop pattern may be found in overweight people and a higher frequency of the ulnar loop pattern may be found in obese people.\n\nThis data may contribute to this field of research and allow better and more adequate referrals possible for people that have a predictive marker of fetal origin of obesity.\n\nAs a limitation, because it was a cross-sectional study, it was not possible to associate the results with important factors, such as prenatal and family history, and it is recommended in future studies that a cohort-type follow-up should be performed to verify a possible association between these factors and the level of physical activity, along with fingerprints.\n\n\nData availability\n\nOpen Science Framework: Data file new 5_Dermatoglyphical impressions are different between children and adolescents with normal weight, overweight and obesity https://doi.org/10.17605/OSF.IO/AFN62 (Alberti, 2019)\n\nThis project contains the following underlying data:\n\n- Data file new 5_Dermatoglyphical impressions are different between children and adolescents with normal weight, overweight and obesity.xlsx (demographic information, the number of finger lines and fingerprint pattern types for 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAlberti A: Data file new 5_Dermatoglyphical impressions are different between children and adolescents with normal weight, overweight and obesity. 2019. http://www.doi.org/10.17605/OSF.IO/AFN62\n\nBabler WJ: Embryologic development of epidermal ridges and their configurations. Birth Defects Orig Artic Ser. 1991; 27(2): 95–112. PubMed Abstract\n\nBhardwaj N, Bhardwaj P, Tewari V, et al.: Dermatoglyphic analysis of fingertip and palmer print patterns of obese children. Int J Med Sci Public Health. 2015; 4(7): 946–949. Publisher Full Text\n\nBhat GM, Mukhdoomi MA, Shah BA, et al.: Dermatoglyphics: in health and disease - a review. J Res Med Sci. 2012; 2(1): 31–37. Publisher Full Text\n\nCummins H, Midlo CH: Finger Prints, Palms and Soles an Introduction to Dermatoglyphics. Dover Publications, inc. New York, 1961. Reference Source\n\nDipietro: Prenatal Development. Encycl Infant Early Child Dev 2. 2008; 4: 359–65.\n\nGarcía-Hermoso A, Correa-Bautista JE, Olloquequi J, et al.: Health-related physical fitness and weight status in 13- to 15-year-old Latino adolescents. A pooled analysis. J Pediatr (Rio J). 2018; 5: pii: S0021-7557(18)30047-0. PubMed Abstract \| Publisher Full Text\n\nGupta A, Karjodkar F: Role of dermatoglyphics as an indicator of precancerous and cancerous lesions of the oral cavity. Contemp Clin Dent. 2013; 4(4): 448–453. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerrera BM, Keildson S, Lindgren CM: Genetics and epigenetics of obesity. Maturitas. 2011; 69(1): 41–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nINEP National Institute of Studies and Educational Research Anísio Teixeira. [date unknown]. Educational indicators. [accessed 2017 July 5]. Reference Source\n\nKing S, Mancini-marie A, Brunet A, et al.: Prenatal maternal stress from a natural disaster predicts dermatoglyphic asymmetry in humans. Dev Psychopathol. 2009; 21(2): 343–353. PubMed Abstract \| Publisher Full Text\n\nMartínez García RM, Jiménez Ortega AI, González Torres H, et al.: Prevention of obesity from perinatal stage. Nutr Hosp. 2017; 34(Suppl 4): 53–57. PubMed Abstract \| Publisher Full Text\n\nMinistério da Saúde: Orientações para coleta e análise de dados antropométricos em serviços de saúde: norma técnica do sistema de vigilância alimentar e nutricional – SISVAN. Brasília, DF: Ministério da Saúde, 2011; 76. Reference Source\n\nMittal VA, Dean DJ, Pelletier A: Dermatoglyphic asymmetries and fronto-striatal dysfunction in young adults reporting non-clinical psychosis. Acta Psychiatr Scand. 2012; 126(4): 290–297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg M, Fleming T, Robinson M, et al.: Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014; 384(9945): 766–781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNodari-junior RJ, Heberle A, Emygdio RF, et al.: Impressões Digitais para Diagnóstico em Saúde: validação de Protótipo de Escaneamento Informatizado. Rev Salud Pública. 2008; 10(5): 767–776. Reference Source\n\nNodari-Júnior RJ, Fin G: Dermatoglifia: impressões digitais como marca genética e de desenvolvimento fetal. Joaçaba: Ed. Unoesc. 2016. Reference Source\n\nOladipo GS, Afolabi EO, Esomunu C: Dermatoglyphic Patterns of Obese versus Normal Weight Nigerian Individuals. J Biomed. 2010; 1: 66–69. Reference Source\n\nOrnellas F, Carapeto PV, Mandarim-de-Lacerda CA, et al.: Obese fathers lead to an altered metabolism and obesity in their children in adulthood: review of experimental and human studies. J Pediatr (Rio J). 2017; 93(6): 551–559. PubMed Abstract \| Publisher Full Text\n\nPasetti SR, Gonçalves A, Padovani CR: Dermatóglífos de mulheres obesas brasileiras. Medicina. 2012; 45(5): 452–459. Publisher Full Text\n\nRaniwala A, Wagh DD, Dixit-Shukla A, et al.: Study and Correlation of Clinical, Radiological, Cytological, and Histopathological Findings in the Diagnosis of Thyroid Swellings. J Datta Meghe Inst Med Sci Univ. 2017; 12(3): 138–142. Publisher Full Text\n\nSharma MK, Sharma H: Dermatoglyphics: A diagnostic tool to predict diabetes. J Clin Diagn Res. 2012; 6(3): 327–332. Reference Source\n\nShetty SS, Johnli AR, Mohd NFB, et al.: Dermatoglyphics: A prediction tool for dental caries. IJDRD. 2016; 4(2): 30–32. Publisher Full Text\n\nWHO - World Health Organization: Physical status: the use and interpretation of anthropometry. Technical Report Series nº 854. Geneva: World Health Organization, 1995; 452. Reference Source\n\nWHO - World Health Organization: World Health Statistics. Geneva: World Health Organization, 2014; 180. Reference Source\n\nWijerathne BT, Meier RJ, Salgado SS, et al.: Dermatoglyphics in kidney diseases: a review. SpringerPlus. 2016; 5(1): 1–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYohannes S: Dermatoglyphic meta-analysis indicates early epigenetic outcomes & possible implications on genomic zygosity in type-2 diabetes [version 1; peer review: 2 approved]. F1000Res. 2015; 4: 617. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "51076", "date": "27 Aug 2019", "name": "Tarimobo Otobo", "expertise": [ "Reviewer Expertise Development and Validation of Outcome measurement tools", "Physical anthropometry", "and big data computational analytics." ], "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 attempted to study and report the finger dermatoglyphic characteristics of normal, overweight and obese children. The choice of cross-sectional study design for the purpose of reporting the palmer dermatoglyphics characteristics in the sample population is commendable as it provided the baseline distribution of arch, whorl ulna, and radial loops in the study population. The analysis of the distribution and comparison of the finger dermatoglyphic characteristics between the normal, overweight and obese subjects was exceptional.\nI am not sure if some of the conclusions were intended, e.g. suggesting that a particular fingerprint pattern is predictive of an outcome (in this case either of obese, overweight and normal weight) was overreaching. More so, since the study design and statistical analysis, did not provide sufficient evidence for such a conclusion.\n\nThe correct reference of the manual ink pad dermatoglyphic collect method is by Antonok et al.1\nIt was challenging to comprehend most of the paragraphs, because of challenging lexical semantics. A detail grammatical edit is recommended.\nIn general, the objective and study design of the article good; however, a major revision is required before final publication release.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "62253", "date": "29 Apr 2020", "name": "Lavanya Prathap", "expertise": [ "Reviewer Expertise Dermatoglyphics", "breast cancer", "epigenetics", "Human anatomy" ], "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 has attempted to associate the genetic and epigenetic influence of obesity and overweight which is represented in dermatoglyphic patterns. The author’s interest in predicting the epigenetic influence of obesity through a non-invasive, cost – effective way of analysis is highly appreciable. The author used dermatoglyphic scanner instead of traditional method for data collection improves the accuracy. The methodology is clear and reproducible. He used appropriate statistical tools and the results conclude significant difference between the obese, overweight and healthy population. Few corrections to be made –\nIntroduction section – last paragraph –last sentence - kindly check the sentence formation.\n\nUnder Methodology - Collection of demographic details - avoid using ‘patient’ can alternate it with ‘participants’.\n\nUnder Results section – Paragraph 1- The description is repeated, can avoid repetition.\n\nUnder results section – Paragraph – 2 - the codes used for digits are varying. Can use any one form of representation either Mesql2 or MET2.\n\nUnder the results section in the description, the digits are represented as MET / But in the reference table, it is represented as Mesql. Kindly fix with one form of representation and use the same in both description and data table.\n\nThe article is recommended for publication after the above said corrections.\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": "64349", "date": "24 Jun 2020", "name": "Ali Azhar Dawasaz", "expertise": [ "Reviewer Expertise Dermatoglyphics", "Radiation hazards", "dentistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 tried to address the relationship between dermatoglyphics and nutritional status (normal, overweight, and obese) in participants aged 10 to 19 years from the center-west region of Santa Catarina, Brazil using BMI. This is a cross-sectional study on a large sample population and addresses an important health parameter like obesity that is not only local but a global phenomenon. Dermatoglyphics is a relatively easier screening tool that is gaining importance in identifying health-related risks. This article is an important addition to the current literature.\nSome concerns however remain and need some additional information from the authors.\nI would like to ask authors to discuss the importance of the use of BMI when there are other more reliable tools to screen individuals’ health statuses. BMI is considered a macro tool and can be used to calculate a baseline value for an ethnically identified population. However, BMI carries inherent flaws when incorporated in a correlation study where a mixed population is under investigation.\n\nIntroduction section, line 6 says: By 2014, more than 1.9 million adults were overweight. Of these, 600 million were obese. Please correct the discrepancy in figures and provide a valid reference.\n\nPage 4, under BMI calculation, the formula needs to be corrected. BMI=Weight in kg/ (Height in mtr)2\n\nPlease provide name and company details of the commercially available dermatoglyphic reader used in this study.\n\nThe weight of participants was noted on a digital scale nonetheless, considering variations caused due to intake of food or water prior to measurement would give varying results and could impact the reliability of data observed. It is requested that the authors kindly add an additional statement regarding the above concern whether taken into consideration or not.\n\nIn general, the objectives and study design are acceptable; however, the conclusions drawn from the study allowing prediction of obesity using DGF remains unclear. Authors are requested to carry out additional statistical tests (Regression/correlation) in order to have a compelling reason to conclude 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?\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": "62596", "date": "01 Jul 2020", "name": "Jannu Chiranjeevi", "expertise": [ "Reviewer Expertise Physical therapy", "Physiotherapy", "Neurosciences", "Rehabilitation", "ergonomics", "stroke", "cerebral palsy", "spinal cord injuries", "diabetes", "diabetic neuropathy" ], "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\nFirstly I congratulate the authors for doing great work on the title \"Dermatoglyphical impressions are different between children and adolescents with normal weight, overweight and obesity: a cross-sectional survey.\"\nIntroduction:\nThe introduction was very clear and the authors have clearly described obesity and dermatoglyphic features in relation to a variety of diseases. The aim of the study was well determined about obesity in children and adolescents aged 10 to 19 years in the centre west region of Santa Catarina.\n\nMethods:\nUnder this section, the study design and study participants were well documented. But I feel that the census was made only between 2013 - 2014. It could be more productive if they have done in at least 5 years census. The collection of demographic characteristics and fingerprint collection and analysis within the schools was done properly by the authors.\n\nStatistical analysis using ANOVA was well apt to the study and the results were well documented under the results section. The table and figures are giving proper information about the study.\n\nDiscussion:\nThe whole study was well summarised under this section. The limitations of the study were well documented.\n\nOverall it's a very good study which is most beneficial.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-964
https://f1000research.com/articles/7-1560/v1	26 Sep 18	{ "type": "Systematic Review", "title": "Pulpotomy versus pulpectomy in the treatment of vital pulp exposure in primary incisors. A systematic review and meta-analysis.", "authors": [ "Lamia Gadallah", "Mahmoud Hamdy", "Adel El Bardissy", "Mohamed Abou El Yazeed", "Mahmoud Hamdy", "Adel El Bardissy", "Mohamed Abou El Yazeed" ], "abstract": "Background: Early childhood caries is a serious public health problem in both developing and industrialized countries. When caries extend to involve the pulp, various forms of pulp treatment are tried to stimulate tooth repair. The choice of proper technique is as important as choosing between different pharmacotherapeutic agents used in treatment of primary teeth. Although pulpotomy is the treatment of choice for vital primary tooth pulp exposure but there is a trend among many dentists to perform pulpectomies for pulp treatment of vital primary anterior teeth. This study aimed to assess the effect of pulpotomy and pulpecomy in treatment of carious vital pulp exposure in primary incisors. Methods: We searched Pubmed and Cochrane library databases up to March, 2018, OpenGrey for grey literature and ClinicalTrials.gov for ongoing trials. Randomized controlled trials comparing between pulpotomy and pulpectomy in treatment of vital pulp exposure in primary incisors were included. Primary outcomes were clinical failure and radiological failure. Data synthesis was performed with a pair wise meta-analysis using fixed-effect models. Results: Four trials were identified for qualitative assessment, only three trials were included in meta-analysis after exclusion of one trial due to its high risk of bias. The pooled results of the longest follow up period for clinical and radiographic failure showed no statistically significant difference between pulpotomy and pulpectomy. The relative risk (RR) was 0.74 with 95% CI 0.46 to 1.21 for radiographic failure and RR 2.69, 95% CI 0.76 to 9.58 for clinical failure. The evidence was limited by the small number of trials included in the meta-analysis. Conclusions: Both pulpotomy and pulpectomy can be used successfully in the treatment of vital pulp exposure in primary incisors. Further high quality studies comparing between pulpotomy and pulpectomy in primary incisors with longer follow up period till exfoliation time.", "keywords": [ "Pulpotomy", "Pulpectomy", "Root Canal Therapy", "Primary Incisor", "Vital Pulp Exposure" ], "content": "Introduction\n\nDental caries is an international public health challenge, especially among young children. Early childhood caries (ECC) is a serious public health problem in both developing and industrialized countries1. ECC has been considered to be at epidemic proportions in the developing world2–6. Treatment of ECC can be accomplished through different types of intervention. When caries extend to involve the pulp, various forms of pulp treatment have been used to treat and/or remove the pulp or to stimulate tooth repair. The choice of technique is as important as the choice between the different pharmacotherapeutic agents which are used in the treatment of primary teeth7.\n\nAlthough pulpotomy is the treatment of choice for vital primary tooth pulp exposure throughout the pediatric dental literature8, the current trend amongst many dentists is to perform pulpectomies for the pulp treatment of carious vital primary anterior teeth9.\n\nThe most common materials used for pulpotomy are formocresol, ferric sulfate, also calcium hydroxide has been used, but with less long term success and more recently mineral trioxide aggregate (MTA) which is much more expensive8,10.\n\nAccording to the latest recommendations of the American Academy of Pediatric Dentistry, both formocresol and MTA are strongly recommended to be used in pulpotomy with moderate quality of evidence. Ferric sulfate, laser and sodium hypochlorite are conditionally recommended but as for calcium hydroxide there is a recommendation against its use in pulpotomy11.\n\nIn pulpectomy a resorbable material such as nonreinforced zinc/oxide eugenol (ZOE), a combination paste of iodoform and calcium hydroxide (Vitapex, Metapex) or a combination paste of zinc oxide and eugenol, iodoform and calcium hydroxide (Endoflas) are used to fill the canals12.\n\nAccording to a Cochrane systematic review there was no conclusive evidence supporting the superiority of one material for use in pulpectomy. Zinc oxide and eugenol, Metapex and Endoflas were found to be equally effective while with low quality of evidence zinc oxide and eugenol may be better than Vitapex but further research is required for confirmation13.\n\nThere are few studies that have compared pulpectomies with pulpotomies in vital primary incisors14. Therefore the claim that pulpotomies don’t work in primary anterior teeth is not supported by high-quality evidence from research. Moreover two studies recently showed that there was no significant difference in success rates of pulpotomies and pulpectomies in the pulp treatment of asymptomatic vital primary incisors 9,15.\n\nWe therefore aimed to determine in patients with carious vital pulp exposure in primary incisors if pulpotomy is better than pulpectomy in terms of pain, soft tissue pathology, tooth mobility, pathological root resorption, periapical radiolucency, pulp canal obliteration and tooth survival based on evidence from randomized controlled trials.\n\n\nMethods\n\nTypes of participants. Children with carious vital pulp exposure in primary incisors.\n\nTypes of interventions. Pulpotomy and pulpectomy (root canal treatment) techniques with different medicaments.\n\nPrimary outcomes. We defined two primary outcomes: clinical failure and radiological failure.\n\nSecondary outcomes. According to the core set of component outcomes as specified by Smaïl-Faugeron et al.16 these secondary outcomes were considered:\n\nSecondary clinical outcomes: pain, soft tissue pathology (gingival swelling, fistulous tract), pathological mobility and tooth survival.\n\nSecondary radiographical outcomes: pathological radiolucency, pathological root resorption, pulp canal obliteration.\n\nRandomized controlled trials comparing pulpotomy and pulpectomy techniques in the treatment of carious vital pulp exposure in primary incisors were included.\n\nElectronic search. We searched the electronic databases as the Cochrane library to 1/3/2018 and Pubmed to 1/3/2018. We developed detailed search strategies for each database searched. We placed no restrictions on the date of publication when searching the electronic databases. The search strategy included appropriate keywords, and Mesh terms when applicable; combined with Boolean operators “AND”, “OR” and “NOT as shown in Table 1 and Table 2. We also searched OpenGrey for grey literature and ClinicalTrials.gov for ongoing trials.\n\nHand searching. We hand searched the reference lists of the included full text articles.\n\nSelection of studies. Two review authors (LG and AEB).independently scanned the titles of all reports identified by the search to determine whether the studies were relevant. They independently scanned selected abstracts to determine whether the study was relevant. After obtaining the full report for all relevant articles, they independently scanned the full reports and completed the data extraction form to determine whether the article should be included or excluded. Disagreements at each stage were resolved by discussion. If this did not resolve the disagreement a third author was invited to settle the disagreement. This did not occur over the course of this study.\n\nData extraction and management. Two review authors (LG and AEB) collected the data independently using a specially designed data extraction form (Dataset 117).\n\nFor each trial, the following data were recorded: the year of publication, the country where the trial took place, detailed description of methodology, sample size, mean age of participants, duration of follow-up and reported outcomes. We contacted the authors of randomized controlled trials for missing information if needed.\n\nAssessment of risk of bias in included studies. Two review authors (LG and AEB) independently assessed the risk of bias in the included trials. The assessment was according to Cochrane risk of bias tool for quality assessment of randomized controlled trials described in the Cochrane Handbook for Systematic Reviews of Interventions 5.1.0 (updated March 2011)18. Any disagreements between the two authors were resolved by discussion. If this did not resolve the disagreement a third author was invited to settle the disagreement. This did not occur over the course of this study.\n\nMeasures of treatment effect. Estimated effect size was calculated as risk ratios with 95% Confidence Interval (CI) for dichotomous outcomes. The unit of analysis was the tooth, because teeth were randomly assigned to intervention.\n\nData synthesis. The effect sizes and associated 95% confidence intervals were calculated using the Mantel-Haenszel method. If the results from trials were homogenous then fixed effect model was preferred. The statistical analysis was performed with the Review Manager program v.5.3 (RevMan)19.\n\n\nResults\n\nBy searching the different databases, 14 references were identified after removal of the duplicates. By scanning the titles and abstracts, 4 studies were included as shown in the Prisma flow diagram Figure 1 and data extraction was performed (completed PRISMA checklist is available as Supplementary File 1).\n\nA list of included articles is shown in Table 3.\n\nYear of publication, setting and operators. The trials were published between 2004 and 2017. Two trials were in Canada by Nguyen et al.15 and Casas et al.21 one trial in the United states of America by Howley et al.9 and one in Iran by Aminabadi et al.20. Operators were dentists in the four trials.\n\nParticipants. The age range of participants with carious vital pulp exposures in primary incisors varied varying from 18 to 60 months.\n\nNumber of arms. All four trials by Nguyen et al.15, Howley et al.9, Aminabadi et al.20 and Casas et al.21 were two-arm studies.\n\nDuration of follow up. Follow up was at 12 and 24 months in two trials, by Aminabadi et al.20 and Casas et al.21. Follow up was to 23 months at three intervals: 5–9, 10–14, and 15–23 months in the trial by Howley et al.9 and at 12 and 18 months in the trial by Nguyen et al.15.\n\nAnesthesia. Three trials, Nguyen et al.15, Howley et al.9 and Casas et al.21, were under general anesthesia and one trial was under local anesthesia, Aminabadi et al.20.\n\nRubber dam. All four trials used rubber dam isolation9,15,20,21.\n\nTreatment. The following treatments were compared in the included trials:\n\nThe Casas et al. trial used ferric sulphate in pulpotomy compared with zinc oxide and eugenol in pulpectomy in 21.\n\nFormocresol in pulpotomy was compared with zinc oxide and eugenol in pulpectomy in the Aminabadi et al. trial20.\n\nFormocresol in pulpotomy was compared with vitapex (calcium hydroxide/ iodoform paste) in pulpectomy in the Howley et al. trial9.\n\nFerric sulfate and mineral trioxide aggregate in pulpotomy was compared with zinc oxide and eugenol in pulpectomy in the Nguyen et al. trial15.\n\nMedicaments\n\nPulpotomy\n\nTwo trials by Howley et al.9 and Aminabadi et al.20 used formocresol to achieve hemostasis, with zinc oxide and eugenol as capping material. One trial used ferric sulphate to achieve hemostasis and zinc oxide and eugenol as capping material, Casas et al.21, and one trial used ferric sulfate to achieve hemostasis, with mineral trioxide aggregate as a capping material, Nguyen et al.15.\n\nPulpectomy\n\nZinc oxide and eugenol was used in three trials by Nguyen et al.15, Aminabadi et al.20 and Casas et al.21. One trial, Howley et al., used vitapex (calcium hydroxide/iodoform paste)9.\n\nPulp access. Following caries removal and pulp exposure, the pulp chamber was accessed using a sterile no. 56 bur in a water-cooled high-speed handpiece and was refined using a sterile round bur in a slow-speed handpiece in three trials, Nguyen et al.15, Aminabadi et al.20 and Casas et al.21.\n\nIn the trial Howley et al.9 the pulp chamber was unroofed using a no. 330 sterile bur in a water-cooled high-speed handpiece then the access was refined using a sterile round bur in a slow-speed handpiece.\n\nPulpotomy. The coronal pulp was amputated using a sharp spoon excavator in two trials by Howley et al.9 and Aminabadi et al.20 and was amputated using a sterile low-speed round bur in another two trials by Nguyen et al.15 and Casas et al.21.\n\nRoot canal treatment. In three trials Nguyen et al.15, Howley et al.9 and Casas et al.21 pulp tissue was removed en bloc using two or more endodontic files (Hedström ﬁles or K files), if the ﬁrst attempt was unsuccessful, the procedure was repeated until all of the pulp tissue was removed.\n\nIn the fourth trial by Aminabadi et al.20 an endodontic K file was introduced to the working length after a periapical radiograph was taken, and most of the pulp tissue was removed completely on the first attempt. If the first attempt was unsuccessful, the procedure was repeated and canals were generally enlarged three sizes past the initial file.\n\nIrrigation. The irrigation solution was sterile water in Nguyen et al. trial15 and saline in the trials by Howley et al.9 and Aminabadi et al.20 while in the fourth trial by Casas et al.21 the irrigating solution was unidentified.\n\nFinal restoration. Resin restorations was performed in three trials, Nguyen et al.15, Aminabadi et al.20 and Casas et al.21. Full coverage crown whether stainless steel crown (SCC) or SCC with white esthetic veneer in Howley et al.9.\n\nNumber of visits. In the four trials by Nguyen et al.15, Howley et al.9, Aminabadi et al.20 and Casas et al.21 the intervention was completed in one session, whether they were performed under general or local anesthesia.\n\nClinical failure. Clinical failure was reported to be 2% and 3% for pulpotomy at 12 months and 18 months respectively, while for the pulpectomy there were none at 12 months, and 1% at the 18 months follow up period in the Nguyen et al. trial15. No clinical failures were reported for neither pulpotomy nor pulpectomy in this 23 month trial by Howley et al.9. The clinical failure rate was 13.1% for pulpotomy and 4.4% at 2 years follow up for Aminabadi et al.20. Clinical failure was 22% in the pulpotomy group, with no clinical failures in the pulpectomy group in the trial by Casas et al.21.\n\nRadiological failure. Radiographical failure was reported to be 3% and 7% in the pulpotomy group at 12 and 18 months respectively, while for the pulpectomy group it was 8% at both 12 months and 18 months follow ups in the Nguyen et al. trial15. Cumulative radiographical results showed failure in 11% in the pulpotomy group, and 27% in pulpectomy group in the Howley et al. trial9. Radiographical failure was 23.9% in the pulpotomy group, and 8.6% in pulpectomy group at 2 years follow up as reported by Aminabadi et al.20. Radiographical failure was in 41% in the pulpotomy group and 18% in pulpectomy group at 2 years follow up trial by Casas et al.21.\n\nOverall failure. One incisor was lost early and counted as a failure in the pulpotomy group in the Howley et al. trial9. One tooth had to be extracted postoperatively (2.2%) in pulpotomy group in the Aminabadi et al. trial20.\n\nPain. One tooth with spontaneous pain (1%) was reported in pulpotomy group in Nguyen et al. trial15. No pain was reported in either group in Howley et al. trial9. Two teeth were reported as having spontaneous pain (4.4%) in the pulpotomy group, and one (2.2%) in the pulpectomy group in Aminabadi et al.20. No pain was reported in either group in the Casas et al. trial21.\n\nSoft tissue pathology. Two teeth showed fistula (2%) in the pulpotomy group, and one tooth had soft tissue swelling (1%) in pulpectomy group in the Nguyen et al. trial15. No soft tissue pathology was reported in either group in Howley et al.9. The presence of fistula was reported in 3 teeth (6.6%) in the pulpotomy group of the Aminabadi et al. trial20. The presence of gingival swelling or parulis in 9 teeth (22%) in pulpotomy group was reported by Casas et al.21.\n\nPathological mobility. Pathologic mobility was not reported for any tooth in three trials, Howley et al.9 Aminabadi et al.20 and Casas et al.21. Only one tooth with pathological mobility was reported by Nguyen et al.15.\n\nPathological radiolucency. The odds ratio for periapical radiolucency was 177.55; 95% CI 20.29 to 1554.01 (P<0.0001; chi-square test) in the Nguyen et al. trial15. After 23 months follow up, 7 teeth (23%) showed frank periapical radiolucency in the pulpectomy group, while only 1 tooth (3%) showed frank periapical radiolucency in the pulpotomy group in Howley et al. trial9. At 2 years follow up 5 teeth (11.11%) showed periapical radiolucency in the pulpotomy group, while only one tooth (2.17%) in the pulpectomy group showed periapical radiolucency in the Aminabadi et al. trial20. At 2 years follow up 7 teeth (58%) showed periapical radiolucency in the pulpotomy group, and 3 teeth (27%) in the pulpectomy group in Casas et al.21.\n\nPathological root resorption. The odds ratio for external root resorption was 136.41;95% CI 15.02 to 1238.27 (P<0.0001; chi-square test) in Nguyen et al. trial15. After 23 months follow up, 2 teeth (7%) showed large external root resorption in the pulpotomy group, and 4 teeth (14%) in the pulpectomy group, while for internal resorption one tooth (3%) showed perforating internal root resorption in the pulpotomy group in the Howley et al. trial9.\n\nAt 2 years follow up pathologic external or internal root resorption occurred in 6 teeth (13.3%) of the pulpotomy group and in 2 teeth (4.34%) of the pulpectomy group in the Aminabadi et al. trial20\n\nAt 2 years follow up pathologic external root resorption occured in 4 teeth (33%) in the pulpotomy group and in 3 teeth (27%) in the pulpectomy group, internal resorption was observed in 17% of the pulpotomy group in Casas et al.trial21\n\nPulp canal obliteration. At 23 months pulp canal obliteration was seen in 18 teeth (60%) in the pulpotomy group in Howley et al.9. At 2 year follow up no teeth showed pulp canal obliteration in Aminabadi et al. trial20. At 2 years follow up 3 teeth (25%) showed pulp canal obliteration in the pulpotomy group in the Casas et al. trial21.\n\nTooth survival. The survival rate was 0.94 (95 % CI equals 0.89 to 0.97) for pulpotomy and 0.97 for pulpectomy at 18 months in Nguyen et al. trial15. The survival rate was 63% for pulpotomy and 85% for pulpectomy at 2 years follow up in Casas et al. trial21.\n\nRisk of bias in included studies. The risk of bias of included studies is shown in Table 4, Table 5, Table 6, and Table 7. The overall risk of bias was low in two trials, Nguyen et al.15 and Aminabadi et al.20. The risk of bias was unclear for clinical assessment in two trials, Howley et al.9 and Casas et al.21, and also for random sequence generation in Casas et al.21. One trial, Casas et al.21, showed high risk of bias due to high percentage of dropped out cases. The risk of bias of included studies is also summarized in Figure 2.\n\nSynthesis of results. Only three trials were included in the meta-analysis by Nguyen et al.15 Howley et al.9 and Aminabadi et al.20 as one trial by Casas et al.21 was excluded due to its high risk of bias. Data were extractable from all three RCTs totaling 338 teeth.\n\nThe data of the longest follow up period was included in the meta-analysis, this was 24 months for Aminabadi et al.20, 18 months for Nguyen et al.15, and at 15 to 23 months for Howley et al.9.\n\nClinical failure\n\nAt the longest follow up period the pooled results showed no statistically significant difference in clinical failure for pulpotomy compared to pulpectomy (RR 2.69 ,95% CI 0.76 to 9.58) as shown in Table 8 and Figure 3.\n\nRadiographic failure\n\nThe pooled results showed no statistically significant difference in radiographical failure for pulpotomy compared to pulpectomy (RR 0.74, 95% CI 0.46 to 1.21) as shown in Table 8 and Figure 4.\n\nPain\n\nThe pooled results showed no statistically significant difference in pain for pulpotomy compared to pulpectomy (RR 2.06, 95% CI 0.31 to13.8)).\n\nSoft tissue pathology\n\nThe pooled results showed no statistically significant difference in soft tissue pathology for pulpotomy compared to pulpectomy (RR 3.11, 95% CI 0.54 to17.7)\n\nPathological radiolucency\n\nThe pooled results of two trials showed no statistically significant difference in periapical radiolucency for pulpotomy compared to pulpectomy (RR 0.75, 95% CI 0.27 to 2.11).\n\nPathological root resorption\n\nThe pooled results of two trials showed no statistically significant difference in pathologic resorption for pulpotomy compared to pulpectomy (RR 1.5, 95% CI 0.56 to 4.04).\n\n\nDiscussion\n\nPulp therapy is performed to preserve primary teeth and maintain its developmental, esthetic, and functional capabilities. Pulpotomy and root canal therapy have been both performed as techniques for the management of asymptomatic vital primary incisors with large carious lesions where removal of caries will lead to pulp exposure20. However the preference of many pediatric dentists to perform pulpectomy in primary incisors was due to the fact that they were taught to do so in their pediatric dentistry residencies and not due to evidence from high quality research9. The aim of this systematic review was to compare between pulpotomy and pulpectomy clinically and radiographically in the treatment of carious vital pulp exposure in primary incisors.\n\nUpon performing our systematic search there were only four randomized controlled trials that have compared pulpotomy and pulpectomy outcomes in vital primary incisors9,15,20,21. After exclusion of one trial due to its high risk of bias that left us three trials to be included in the meta-analysis. The data of the longest follow up period was included as the follow ups were close to each other ranging from 15 months to 24 months and they best reveal the efficacy of the performed techniques.\n\nThe results were calculated with risk ratio (RR) effect measure and confidence intervals (CIs). The pooled results of the clinical and radiographical failure as the primary outcome showed no statistically significant difference between pulpotomy and pulpectomy, although the relative risk RR was 0.74 with 95% CI 0.46 to 1.21 which means lower risk for radiographic failure in pulpotomy than pulpectomy and the RR was 2.69 with 95% CI from 0.76 to 9.58 for clinical failure which means higher risk for clinical failure in pulpotomy rather than pulpectomy but all the outcomes showed CIs including the number 1 that means that there was no statistical significant difference between pulpotomy and pulpectomy cases.\n\nFor the clinical outcomes pain and soft tissue pathology, the pooled results for these outcomes showed no statistically significant difference between pulpotomy and pulpectomy while pathologic mobility was only reported for one incisor in one trial.\n\nThe radiographic outcomes included periapical radiolucency and pathologic root resorption, the pooled results also showed no statistically significant difference between pulpotomy and pulpectomy. We considered the tooth to be scored with pathologic root resorption if it showed perforating internal root resorption or large external root resportion while those teeth showing contained internal root resorption or questionable external root resorption were not counted. For periapical radiolucency, only frank radiolucencies were counted and not questionable ones.\n\nPediatric dentists do not consider the radiographic pathological changes as questionable radiolucencies or limited pathological root resorptions to be an absolute indication for extraction taking into consideration the absence of clinical pathological signs or symptoms15.\n\nOverall failure was reported for two pulpotomized incisors in two trials. Regarding tooth survival although it is an important outcome but it is not commonly reported.\n\nThis study showed that there is no statistical significant difference in clinical and radiographical success rates of pulpotomy and pulpectomy with different medicaments in the treatment of carious vital pulp exposure in primary incisors and this refutes the misconception among some pediatric dentists that pulpotomy does not work in primary incisors.\n\nThe evidence was limited by the small number of trials included in the meta-analysis. The overall risk of bias of primary studies was low for three trials, except for the unclear risk for blinding of clinical assessment which was not effective, we did not have access to all the trial protocols to assess the selective reporting bias except only one trial.\n\n\nConclusion\n\nThere was no statistical significant difference in the success rates of pulpotomies and pulpectomies in the pulp treatment of carious vital pulp exposure in primary incisors. We recommend teaching of pulpotomy as a treatment option for vital pulp exposure in primary incisors in pediatric dentistry residency programs and further high quality studies comparing between pulpotomy and pulpectomy in primary incisors with a longer follow up period till exfoliation time.\n\n\nData availability\n\nDataset 1: Summarized extracted data from included trials. 10.5256/f1000research.16142.d21885717", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAuthor information\n\nLG is a Researcher Assistant at the Orthodontics and Pediatric Dentistry Department, National Research Centre, Egypt. MH is a Professor at the Pediatric Dentistry and Dental Public Health Department, Faculty of Dentistry, Cairo University, Egypt. AEB is an Associate Professor at the Pediatric Dentistry and Dental Public Health, Faculty of Dentistry, Cairo University, Egypt. MAEY is a Professor at the Orthodontics and Pediatric Dentistry Department, National Research Centre, Egypt.\n\n\nReferences\n\nColak H, Dülgergil CT, Dalli M, et al.: Early childhood caries update: A review of causes, diagnoses, and treatments. J Nat Sci Biol Med. 2013; 4(1): 29–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilnes AR: Description and epidemiology of nursing caries. J Public Health Dent. 1996; 56(1): 38–50. PubMed Abstract \| Publisher Full Text\n\nSingh S, Vijayakumar N, Priyadarshini HR, et al.: Prevalence of early childhood caries among 3-5 year old pre-schoolers in schools of Marathahalli, Bangalore. Dent Res J (Isfahan). 2012; 9(6): 710–4. PubMed Abstract \| Free Full Text\n\nEl-Yazeed MA, Rashed M, El sayed M, et al.: Dental Caries Prevalence among a group of Egyptian Nurseries Children. Life Sci J. 2011; 8(1): 412–419. Publisher Full Text\n\nAlkhtib A, Ghanim A, Temple-Smith M, et al.: Prevalence of early childhood caries and enamel defects in four and five-year old Qatari preschool children. BMC Oral Health. 2016; 16(1): 73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMothupi KA, Nqcobo CB, Yengopal V: Prevalence of Early Childhood Caries Among Preschool Children in Johannesburg, South Africa. J Dent Child (Chic). 2016; 83(2): 83–7. PubMed Abstract\n\nHugar SM, Deshpande SD: Comparative investigation of clinical/radiographical signs of mineral trioxide aggregate and formocresol on pulpotomized primary molars. Contemp Clin Dent. 2010; 1(3): 146–151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAAPD: Guideline on Pulp Therapy for Primary and Immature Permanent Teeth. In REFERENCE MANUAL. 2014. Reference Source\n\nHowley B, Seale NS, McWhorter AG, et al.: Pulpotomy versus pulpectomy for carious vital primary incisors: randomized controlled trial. Pediatr Dent. 2012; 34(5): 112–9. PubMed Abstract\n\nWalker LA, Sanders BJ, Jones JE, et al.: Current trends in pulp therapy: a survey analyzing pulpotomy techniques taught in pediatric dental residency programs. J Dent Child (Chic). 2013; 80(1): 31–5. PubMed Abstract\n\nAAPD: Use of Vital Pulp Therapies in Primary Teeth with Deep Caries Lesions. REFERENCE MANUAL. 2017; 39(6): 173–186. Reference Source\n\nSubramaniam P, Gilhotra K: Endoflas, zinc oxide eugenol and metapex as root canal filling materials in primary molars--a comparative clinical study. J Clin Pediatr Dent. 2011; 35(4): 365–9. PubMed Abstract \| Publisher Full Text\n\nSmaïl-Faugeron V, Glenny AM, Courson F, et al.: Pulp treatment for extensive decay in primary teeth. Cochrane Database Syst Rev. 2018; 5: CD003220. PubMed Abstract \| Publisher Full Text\n\nSmaïl-Faugeron V, Courson F, Durieux P, et al.: Pulp treatment for extensive decay in primary teeth. Cochrane Database Syst Rev. 2014; (8): CD003220. PubMed Abstract \| Publisher Full Text\n\nNguyen TD, Judd PL, Barrett EJ, et al.: Comparison of Ferric Sulfate Combined Mineral Trioxide Aggregate Pulpotomy and Zinc Oxide Eugenol Pulpectomy of Primary Maxillary Incisors: An 18-month Randomized, Controlled Trial. Pediatr Dent. 2017; 39(1): 34–38. PubMed Abstract\n\nSmaïl-Faugeron V, Fron Chabouis H, Durieux P, et al.: Development of a core set of outcomes for randomized controlled trials with multiple outcomes--example of pulp treatments of primary teeth for extensive decay in children. PLoS One. 2013; 8(1): e51908. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGadallah L, Hamdy M, El Bardissy A, et al.: Dataset 1 in: Pulpotomy versus pulpectomy in the treatment of vital pulp exposure in primary incisors. A systematic review and meta-analysis. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16142.d218857\n\nCochrane Handbook for Systematic Reviews of Interventions. G.S. Higgins JPT, Editor. The Cochrane Collaboration. 2011. Reference Source\n\nReview Manager (RevMan) [Computer program]. The Nordic Cochrane Centre, The Cochrane Collaboration. 2014. Reference Source\n\nAminabadi NA, Farahani RM, Gajan EB: A clinical study of formocresol pulpotomy versus root canal therapy of vital primary incisors. J Clin Pediatr Dent. 2008; 32(3): 211–4. PubMed Abstract \| Publisher Full Text\n\nCasas MJ, Kenny DJ, Johnston DH, et al.: Outcomes of vital primary incisor ferric sulfate pulpotomy and root canal therapy. J Can Dent Assoc. 2004; 70(1): 34–8. PubMed Abstract" }	[ { "id": "39650", "date": "07 Nov 2018", "name": "Mariem O. Wassel", "expertise": [ "Reviewer Expertise pediatric dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe systematic review and meta-analysis regarding pulpotomy versus pulpectomy in the treatment of vital pulp exposure in primary incisors is well designed and that the methodology and discussion are well written. One question I have is: was the protocol of this review registered before carrying out the research? Some mistakes in writing the manuscript are present which are:\nPlease be sure the reference number is written right after the author name through-out the whole manuscript. In the background section of the abstract: This study aimed to assess the (effectiveness) of pulpotomy and pulpecomy (pulpectomy) in treatment of carious vital pulp exposure in primary incisors. In the conclusion section of the abstract; Further high quality studies comparing between pulpotomy and pulpectomy in primary incisors with longer follow up period till exfoliation time\n\n(are needed). Page 2, introduction section:\nIn pulpectomy(,) a resorbable material such as nonreinforced According to a Cochrane systematic review(,) there was no Endoflas were found to be equally effective(.) (W)hile(,) with (a) low quality of evidence(,) zinc oxide and eugenol may be better than We(,) therefore(,) aimed to determine in patients with carious vital pulp exposure in primary incisors(,) if pulpotomy is better than\n\nPage 6:\nDuration of follow up.: “Follow up was at 12 and 24 months in two trials, by Aminabadi et al[ref-1] and Casas et al2.” .do you mean that both studies used a 12 and 24 months follow up period or that the first study used 12 months follow up while the second was 24 months? Please clarify. Follow up was (up) to 23 months at three intervals: 5–9, 10–14, and 15–23 months Anesthesia: under local anesthesia (by) Aminabadi et al1. Medicaments -- Pulpotomy: please rewrite this paragraph noting that 2 trials performed formocresol pulpotomy as we don’t achieve hemostasis with formocresol. Pulp access: In the trial (by) Howley et al3 the pulp chamber was unroofed using Pulpotomy: in another (the other) two trials by Nguyen et al[ref-4] and Casas et al[ref-2]. Final restoration : Resin restorations was (were) performed in three….. Full coverage crowns whether stainless steel crown (SCC) or SCC with white esthetic veneer( were used ) in Howley et al( trial )3. Results of studies; nor pulpectomy in this (the) 23 month(s) trial by Howley et al3. The clinical failure rate was 13.1% for pulpotomy and 4.4% (for pulpectomy) at\n\npage 7:\n\nradiological failure.: was in (remove \"in\") 41% in the pulpotomy group and 18% in pulpectomy group at 2 years follow up trial by Casas et al[ref-2]. Pain: No pain was reported in either group(s) in Howley et al3. trial9.\n\nNo pain was reported in either group(s) in the Casas et al2. trial21. Soft tissue pathology: No soft tissue pathology was reported in either group(s) in Howley et al3. (trial)9.\n\nPathological mobility: Pathologic mobility was not reported for any tooth in three trials (;) Howley et al3.9(,) ,Aminabadi et al1.20 and Casas et al2.21.\n\nPathological radiolucency:\nAt 2 years follow up(,) 5 teeth (11.11%) showed follow up(,) 7 teeth (58%) showed periapical radiolucency in the\n\npathological root resorption: while for internal resorption(,) one tooth (3%) showed perforating\n\nAt 2 years follow up(,) pathologic external or internal root resorption occurred in 6 teeth (13.3%) (in) the pulpotomy group and in 2 teeth (4.34%) (in) the pulpectomy group in the Aminabadi et al. trial20 At 2 years follow up(,) pathologic external root resorption occurred\n\nPulp canal obliteration:\nAt 23 months(,) pulp canal obliterationmwas seen in 18 teeth (60%) in the pulpotomy group in Howley et al.9 (trial). At 2 year follow up(,) no teeth showed pulp canal obliteration\n\nPages 8 and 9:\nPlease add reference number to authors names in the titles of tables 4 to 7.\nPage 11, discussion section:\n2nd paragraph: After exclusion of one trial due to its high risk of bias(,) (only) three trials (were left) to be included in the meta-analysis.(were left) to be included in the meta-analysis. Overall failure was reported for two pulpotomized incisors in two trials. (on the other hand,)\n\ntooth survival(,) although it is an important outcome(,) but it is not commonly reported. The overall risk of bias of primary studies was low for three trials, except for the unclear risk for blinding of clinical assessment which was not effective(.)(W)e did not have access to all the trial protocols to assess the selective reporting bias except (for) only one trial.\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": "4206", "date": "08 Nov 2018", "name": "Lamia Gadallah", "role": "Author Response", "response": "Thank you Dr. Mariem very much for reviewing our study. As for the protocol registration, yes it was registered in the Evidence based Committee of Cairo University as the preliminary step of my Ph.D. in 2014." } ] }, { "id": "41014", "date": "03 Dec 2018", "name": "Armin Shirvani", "expertise": [ "Reviewer Expertise research methodology and statistical 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 manuscript is well structured and the format of the report on the details of the study is acceptable. There are two points to consider in the Meta-analysis:\nThe results of Hawley’s study should not have been removed because of zero in nominators. They could use 0.01 instead of 0 and continue the analysis. Heterogeneity in time intervals is an important issue and if a significant heterogeneity has been detected the results of the meta-analysis would not be valid.\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? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "4292", "date": "20 Dec 2018", "name": "Lamia Gadallah", "role": "Author Response", "response": "Dear Dr. Armin, Many thanks for reviewing our study and for your valuable comments.Concerning your first comment on the exclusion of Howley et al. study from the clinical failure meta-analysis, we returned to Cochrane handbook for systematic reviews of \"intervention that stated that\". The standard practice in meta-analysis of odds ratios and risk ratios is to exclude studies from meta-analysis where there are no events in both arms. This is because such studies do not provide any indication of either the direction or magnitude of the relative treatment effect\"Actually the Revman program that we used for meta-analysis does not accept any decimals in the events in risk ratio as dichotomous outcomes.As for the heterogeneity in the radiographic evaluation, we performed a sensitivity analysis and it was due to one outlying study so we performed the meta-analysis both with and without this study and we stated that the results should be interpreted with caution." } ] } ]	1	https://f1000research.com/articles/7-1560
https://f1000research.com/articles/8-962/v1	25 Jun 19	{ "type": "Research Article", "title": "Accumulation Bias in meta-analysis: the need to consider time in error control", "authors": [ "Judith ter Schure", "Peter Grünwald", "Peter Grünwald" ], "abstract": "Studies accumulate over time and meta-analyses are mainly retrospective. These two characteristics introduce dependencies between the analysis time, at which a series of studies is up for meta-analysis, and results within the series. Dependencies introduce bias — Accumulation Bias — and invalidate the sampling distribution assumed for p-value tests, thus inflating type-I errors. But dependencies are also inevitable, since for science to accumulate efficiently, new research needs to be informed by past results. Here, we investigate various ways in which time influences error control in meta-analysis testing. We introduce an Accumulation Bias Framework that allows us to model a wide variety of practically occurring dependencies including study series accumulation, meta-analysis timing, and approaches to multiple testing in living systematic reviews. The strength of this framework is that it shows how all dependencies affect p-value-based tests in a similar manner. This leads to two main conclusions. First, Accumulation Bias is inevitable, and even if it can be approximated and accounted for, no valid p-value tests can be constructed. Second, tests based on likelihood ratios withstand Accumulation Bias: they provide bounds on error probabilities that remain valid despite the bias. We leave the reader with a choice between two proposals to consider time in error control: either treat individual (primary) studies and meta-analyses as two separate worlds — each with their own timing — or integrate individual studies in the meta-analysis world. Taking up likelihood ratios in either approach allows for valid tests that relate well to the accumulating nature of scientific knowledge. Likelihood ratios can be interpreted as betting profits, earned in previous studies and invested in new ones, while the meta-analyst is allowed to cash out at any time and advice against future studies.", "keywords": [ "meta-analysis", "accumulation bias", "sequential", "cumulative", "living systematic reviews", "likelihood ratio", "research waste", "evidence-based research" ], "content": "PDF\n\n\n\nThe PDF of this article can be downloaded from here.", "appendix": "" }	[ { "id": "50370", "date": "14 Aug 2019", "name": "Steven P. Ellis", "expertise": [ "Reviewer Expertise multivariate analysis", "statistical computing", "applications of topology to data analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article explains how bias arises in meta-analysis and explains how, in theory, one can nonetheless control the error rate through use of the likelihood ratio statistic. I found the use of the likelihood ratio quite interesting.\n2.2, p. 4 \"This is also the case when the timing of the meta-analysis is based on an (overly) optimistic last study estimate or an (overly) optimistic meta-analysis synthesis is considered the final one.\"\nSection 3, p. 4. \"We denote this example ...\" How about \"We CALL this example ...\"\n(3.1a), p. 5: I think it should just be ni, not √ni. [Maybe not?]\np. 5: \"... if the study shows a significant positive effect\" So you are doing one-sided tests. Why do you do alpha/2-size tests, for example 0.025? Why not alpha-size, for example 0.05? Getting a significantly low Z can have the interpretation that that treatment being tested seems to actually be harmful. Therefore no further studies should be done with it.\np. 5: Why is T ≥ t + 1? Why might the current study ultimately prove to be the last one?\np. 6: It seems that the P in (3.2) has nothing to do with the null and alternative hypotheses. That P has to do with the behavior of researchers.\np. 6: The rate 2.5/(2.5+1.9) might be justified by observing that that number is just the conditional probability of getting a positive finding conditional on another study being done.\np. 6: \"As a result, study series that contain more significant studies have larger probabilities to come into existence than those that contain less.\" That sentence is vague.\np. 6: There appears to be a typographical error in formula (3.3). The factors alpha/2 and (1-alpha) shouldn't be in the numerator. However, the value 0.487 on the right-hand side is correct. (Checked by simulation.)\np. 6: \"... the last study is unbiased ...\" What do you mean by \"last study\", the Tth study? The last study before what? Let S denote the number of studies available at the time of the meta-analysis. S is random. But the Sth study is not unbiased (given that there will be a meta-analysis) because the decision to do the meta-analysis partly depends on the outcome of study S. Since the subject of the paper is meta-analysis, it is the first S studies, i.e., the studies available at the time of the meta-analysis, that are relevant.\nSuppose U is a fixed or random time that is statistically independent of the study series. Is the last study before U unbiased? What if the last study before U was published 50 years ago? The fact that no study has been done in 50 years probably says something about the outcome of the last study. So even though U is independent of the study series, conditional on the event that no study has been done in the last 50 years prior to U, that last study is not unbiased. (Perhaps one should pay attention not just to the number of studies that have been performed but also to when they were performed.) It is very difficult to identify a study that is unbiased conditional on everything one knows about the study series. Instead of looking at past studies, one could look at a future study. One might say \"I will start a meta-analysis after the next study is completed\". The next study would be unbiased, but what if there are no further studies? That strategy would work if one knew for sure that there will be a study. For example, if you knew at time U that some researchers have already started -- but not completed -- a study.\np. 7: \"inflation actual inflation\" looks like a typo.\np. 7: Equation (3.5). Have you defined the P̃0 notation? I think you should one sided-tests. (See above.)\np. 8: Probability Notation is a conditional probability. The notation “z1,…,Zt” for a study series apparently hasn't been introduced yet.\np. 9: I don't understand the footnote.\nP. 10: I don't understand the sentence \"In this section we assume that the timing of the meta-analysis test is independent from the estimates that determined the size of the series.\"\np. 15: \"But this effects will be ...\" Perhaps this should be \"But THESE effects will be ...\".\np. 16: \"... which has the same meaning as the fraction ...\" Perhaps this should be \"... which has the same meaning as the RATIO ...\"\nP. 16: \"The likelihood ratio is a test statistic that depends on the specification ...\" Perhaps this should be \"The likelihood ratio is a test statistic that ONLY depends on the specification ...\"\np. 16: \"Any data sampled from an alternative distribution will have the same analysis time probabilities as data ...\" I prefer \"GIVEN THE DATA, any data sampled from an alternative distribution will have the same analysis time probabilities as data ...\"\np. 17: The authors sometimes introduce symbols without defining them. For example, I couldn't find any place where the symbol γ is defined. From context one can figure out what it means, but I would prefer if it were defined somewhere.\np. 19: \"In contrast to an all-or-nothing test for one study, inspecting the betting profit of a study is a way to test the data without loosing the ability...\" I think it should be \"In contrast to an all-or-nothing test for one study, inspecting the betting profit of a study is a way to test the data without LOSING the ability...\".\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": [] }, { "id": "53065", "date": "16 Oct 2019", "name": "Joanna IntHout", "expertise": [ "Reviewer Expertise Biostatistics (i.e. statistician in medical field)", "with an emphasis on meta-analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper explains how bias arises in meta-analysis, as studies never are a random sample and the timing of a meta-analysis neither is random. Timing of the meta-analysis – if performed – and the results of the studies are obviously dependent, resulting in accumulation bias and inflated type I errors. The parallel between conducting studies and betting while continuously reinvesting the profits is an intriguing one.\nI apologize for the long duration of my peer review: the paper was rather intense and long, and sometimes a bit difficult to follow, due to the condensed writing style. It might benefit from some extra sentences and some additional, concrete examples. And, to my opinion the content might also be suitable for a few papers.\nHowever, I could find only minor editorial issues, and I congratulate the authors with this well-written, very relevant paper.\n\nPage 1, abstract\nIt feels a bit like a contradiction when is stated (halfway): “…, no valid p-value test can be constructed. Second, tests based on likelihood ratios withstand Acc. Bias: they provide bounds on error probabilities that remain valid despite the bias.” Also last paragraph: Taking up likelihood ratios… allows for valid tests.\nProbably it has to do with the term p-value based statistical tests that I am not familiar with (as opposed to likelihood ratio tests). I think it has to do with the explanation in the discussion, that (p-value) tail area approaches to testing are very sensitive to the shape of the sampling distribution, but in the abstract, this was not clear to me.\n\n1. Introduction I like how you start with the two quotes. First column at the end: knowledge and all decisionS made along the way. (s is missing)\n\n2. Accumulation Bias Somewhat difficult (although correct) sentence: The crucial point is that not all pilot studies or small study series will reach a meaningful size and that doing so might depend on results in the series. One but last sentence: So meta-analysis also report…. (should be meta-analyses)\n\nSection 3.6\nTypo: The inflation actual inflation in the type-I error… Figure 1: in black and white print the colours in the legend seem to differ from the colours in the graph. Table 2: If possible, it might be handy to add the P̃0 and P0 to the title (after bias only and after bias as well as impaired sampling distribution).\n\nSection 4.5 Typo: These cumulative meta-analysis judge…. : should be meta-analyses\n\nSection 5 Here you refer to “another illustration”… as the Toy Story Scenario. However, where do you discuss this scenario?\n\nSection 6.1\nTypo: “But this effects…”. Also somewhat unclear sentence. I suppose that you mean that is difficult to define a cut-off for the number of early studies to be excluded from meta-analysis.\n\nSection 6.2 Here you compare the prior odds of Ioannidis with the fraction of pilot studies from the null and alternative distribution π / (1-π). However, you did not define π before, and if I understand it correctly, π is the fraction of studies from the alternative distribution, although this text (first line) suggests the other way around.\n\nSection 7.1 Formula 7.2: Please define H1 and H0. Please edit sentence directly below formula: …16 times as many true rejections than (as?) false rejections (with?) γ = (1-β) / α. Formula 7.4: P0 : should be integral over z1, …, zt (instead of z2) (twice). Should it not contain also dz(1).…dz(t)?\nI noticed that you don’t use the word test, only “error control”. It is not fully clear to me: if we use the threshold based on the Bayes posterior odds, does that also result in a p-value, or is it just a yes/no answer? Or can we use a distribution? (you elaborate on this only in Section 9).\n\nAnd how do we specify R= π / (1-π)? Should this be influenced by the study results seen thus far? As you state in section 6.2 on π / (1-π): “the fraction of pilot studies from the null and alternative distribution. … The only available source of information would be previous study results… “. However, this would mean that – indeed - depending on the timing of the meta-analysis, we would define a different R. Or should we use the same – more general - R as Ioannidis, ie 1:1, or 2:1?\n\nInteresting is also that the threshold, that is based on the pre-experimental rejection odds, becomes more stringent if we believe the ratio of true to false rejections to be higher, e.g. if R =2 and γ = 16, the threshold becomes 32, but if R=1, the threshold is 16. Could you elaborate on that? You do elaborate a bit in Section 8, but for me it is still not very clear.\n\nSection 9 Edit, 2nd paragraph, … a series of bets s(Z1), … against the null hypothesis that make a profit…. I suggest to (re)move “against the null hypothesis” to facilitate easier reading. Typo: each null result might costs 1 dollar (should be: cost) “If you cash out, your profit is s(Z1) s(Z2) s(Z3)”. Are the s(Z) bets not odds or probabilities? Should we not add the profit here? And subtract the 1 dollar initial investment? Or do I show here my lack of knowledge on gambling? Only later, when you suggest s(Z) = f1/f0, this makes more sense. Typo, second column before quote of Shafer: twice “under the null”. Inspecting the betting profit of a study: do you mean calculating the LR for that study? I don’t understand the following sentence: “The LR has the ability to maximize the rate of growth among all studies in a series”.\n\nSection 10 Typo: 3rd paragraph: separate meta-analysis of various sizes…, should be meta-analyses\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/8-962
https://f1000research.com/articles/8-959/v1	24 Jun 19	{ "type": "Study Protocol", "title": "Protocol for a phase I trial of a novel synthetic polymer nerve conduit 'Polynerve' in participants with sensory digital nerve injury (UMANC)", "authors": [ "Ralph Murphy", "Alessandro Faroni", "Jason Wong", "Adam Reid", "Ralph Murphy", "Alessandro Faroni", "Jason Wong" ], "abstract": "Background: Peripheral nerve injuries are common, with approximately 9,000 cases in the UK annually. Young working individuals are predominantly affected, leading to significant health and social implications. Functional recovery is often poor with impaired hand sensation, reduced motor function and pain and cold intolerance. Where a nerve gap exists, nerve grafting remains the gold-standard treatment but creates a second surgical site, sensory deficit at the donor site, possible neuroma formation and has limited availability. Current commercially available synthetic and resorbable nerve conduit alternatives are reported to be rigid and inflexible. This study will set out to examine the first-in-man use of a new nerve conduit device ‘Polynerve’ to repair small nerve gaps in digital sensory nerves of the hand. Polynerve is a degradable co-polymer of poly-ε-caprolactone and poly-l-lactic acid, which is shaped as a cylinder that has greater tensile strength, flexibility and less acidic degradation compared with current commercially available synthetic nerve conduits. In addition, it has a novel micro-grooved internal lumen that aids Schwann cell ingress and alignment to improve nerve regeneration. Methods: In total, 17 eligible participants will be recruited to undergo repair of a transected sensory nerve of the hand using the Polynerve device. All participants that receive the nerve conduit device will be followed for a period of 12 months post-surgery. The primary endpoint is safety of the device and the secondary endpoint is degree of sensory nerve regeneration through the conduit assessed using standard sensory testing (2-PD, WEST monofilament testing and locognosia). Discussion: The ‘UMANC’ trial is a single-centre UK-based, prospective, unblinded, phase I clinical trial of a novel nerve conduit device. We aim to demonstrate the safety of Polynerve as a synthetic, biodegradable nerve conduit and improve the treatment options available to patients with significant nerve injuries. Registration: Clinicaltrials.gov: NCT02970864; EudraCT: 2016-001667-37.", "keywords": [ "Peripheral nerve injury", "nerve conduit", "biomaterials", "Poly-ε-caprolactone", "Poly-L-lactic acid" ], "content": "Introduction\n\nPeripheral nerve injuries are a common occurrence, with approximately 9000 cases in the UK occurring each year. Most are in a predominantly young and working population. Where surgical reconstruction is required to repair the peripheral nerve injury, techniques employed have changed little in the last 50–60 years1 with many factors influencing the outcomes, such as age of patient, timing, level and extent of injury, method of repair and the surgeon’s skill2.\n\nDespite advances in microsurgical nerve repair techniques, functional recovery is often poor, and can result in impaired hand sensation, reduced motor function and frequent pain and cold intolerance. This can have a profound and permanent impact on the patient’s recovery and subsequent quality of life. Nerve repair has significant health, social and cost implications with the treatment and rehabilitation of an employed person, estimated to be €51,2383.\n\nPeripheral nerve injury usually presents with nerve stumps that can be approximated in surgical repair: direct, end-to-end suture repair of the epineurium (neurorrhaphy). Excessive tension over the suture line leads to poor results4; therefore, when the nerve stumps cannot be approximated without tension, an alternative surgical method is required.\n\nWhere the nerve gap exceeds more than 5 mm, there are two fundamental options, either ‘nerve grafting’ or ‘tubulisation’ using a bridging material5. This study will examine the first-in-man use of a new nerve conduit device ‘Polynerve’ to repair small nerve gaps in digital sensory nerves of the hand. Polynerve is a degradable co-polymer of poly-ε-caprolactone (PCL) and Poly-L-lactic acid (PLLA) which is shaped as a cylinder with a novel internal lumen consisting of a specific micro-grooved architecture.\n\nIn the presence of a nerve gap, the gold standard is to use the patients’ own nerve tissue (autograft) to bridge the gap in the damaged/severed nerve. The sural/antebrachial cutaneous/posterior interosseus nerves are favourable and commonly used donor nerves to use in such repair. As the harvested nerve undergoes Wallerian degeneration, the autograft functions as a guide or scaffold, with the advantage of the presence of Schwann cells creating a conducive environment for regenerating axons1.\n\nHowever, there are inherent problems in this approach: a second surgical site and sensory deficit at the donor site, limitation of material, possible neuroma formation and graft mismatch5–8.\n\nThis has prompted research on the use of bio-engineered nerve conduits as an artificial means of guiding axonal regeneration. These conduits function as a guide for axonal growth and help maintain an internal environment for nerve regeneration. Currently available nerve conduits are not ideal: several are prepared from inert non-biodegradable materials or from natural collagen with inherent risks of disease transmission and immuno-rejection. Furthermore, the high cost of these devices limits their appeal to the United Kingdom’s National Health Service, where cost-effectiveness is paramount. Despite advances in bioengineering, commercially available conduits to date have not been able to match the results of the current clinical gold standard, i.e. that of reconstruction using a nerve graft.\n\nNerve repair by tubulisation, in which the opposing nerve stumps of a transected nerve are enclosed in a tube (nerve guide conduit), was first described in 1880 when a decalcified bone segment was used to provide a pathway between the ends of a severed nerve9. The tubulisation technique is designed to protect the nerve from the surrounding tissue and potential scar tissue, as well as providing mechanical support, directional guidance for axonal sprouting and an environment permissive to nerve development and regeneration2,10. A number of reviews have been published in which the ideal features of a nerve guide conduit are considered; that is, readily available in appropriate sizes, sterilisable, easily implantable, biocompatible, and with appropriate physical and mechanical characteristics to be flexible and suturable, yet able to withstand compression or collapse2,11.\n\nThe first generation of artificial nerve guide conduits for clinical application were non-resorbable silicone tubes however in many cases further surgeries were required to remove the tubes following nerve regeneration due to the risk of nerve compression6,12. Although silicone and expanded polytetrafluroethylene (ePTFE) have both been used clinically for nerve repair, use of non-resorbable materials in this indication has been associated with chronic nerve compression, decreased axonal conduction and fibrosis8,11 as well as presenting a risk of chronic foreign body reaction with excessive scar tissue formation7.\n\nAs a result of the problems experienced, use of non-resorbable materials in nerve guide conduits has significantly declined. Furthermore, in describing the design criteria for nerve guidance conduits the FDA, who have responsibility for regulatory approval of medical devices for use in the USA, have stated that the material used must be biodegradable10. Conduit dimensions are also highlighted as a critical design feature as diameter and wall thickness are believed to influence the rate of regeneration as well as potentially compressing the growing nerve if the diameter is inadequate10.\n\nResorbable nerve guidance conduit devices may be derived from natural or synthetic materials. Natural polymer-based devices are predominantly derived from collagen because of the inherent biocompatibility and relative abundance of this protein13. A number of Type I bovine collagen derived nerve guidance conduits and wraps have gained regulatory approval for use in peripheral nerve injury: i.e. NeuraGen® (Integra, FDA 510 (k) approval 2001; CE mark 2003), NeuroMatrix®, and NeuroFlex® (Stryker, FDA 510(k) approval 2014)10. In vivo studies with NeuraGen® demonstrated equivalent functional performance in entubulation repair compared to use of standard nerve autograft and direct suture repair14. NeuraGen® clinical data from several prospective and retrospective studies have been published demonstrating clinical safety and efficacy15–17. Although there appeared to be acceptable outcomes in small sensory nerve repair, mixed motor/sensory nerves fared comparatively poorly18.\n\nSynthetic biodegradable polymers offer many advantages in the fabrication of devices for peripheral nerve injury. The materials are readily available and can be engineered to modify physical and mechanical characteristics, such as strength, permeability and degradation rate as well as cell attachment and proliferation by using physical or chemical modifications12.\n\nPolyglycolic acid conduits were the first to be used in clinical studies for peripheral nerve repair7. There is preclinical and clinical data for Neurotube® (Synovis, CE mark 1995; FDA 510 (k) 1999) demonstrating efficacy in gap defects in animal models up to 30 mm and comparable efficacy to primary or nerve graft repair in randomised clinical trials19. Although most safety and efficacy data exist for Neurotube® compared to other nerve graft conduits, the rapid degradation, concomitant reduction in mechanical strength and acidic degradation products are limiting factors to its use10.\n\nPLA and PCL are biocompatible and approved for use in numerous biomedical applications. Polylactide (L- and DL- forms) copolymerised with PCL have found utility in nerve guide conduits and Neurolac® (Polyganics, FDA 510 (k) 2003; CE mark 2004), consists of a poly (65/35(85/15 L/D) lactide ε-caprolactone) phospho-ester. Neurolac® has extensive pre-clinical in vivo data, and in randomised clinical trials Neurolac® is reported to have comparable efficacy to gold standard autograft in defects up to 20 mm20. Known adverse events associated with the use of a Neurolac® nerve guide include but are not limited to: failure to provide adequate nerve regeneration at sites where too much tension or compression occurs; failure to provide adequate/complete nerve regeneration; transitory local irritation; infection; allergy and delayed wound healing. Limitations are reported to be the high rigidity and inflexibility of the device. In comparison to Neurolac®, Polynerve has greater tensile strength, flexibility and less acidic degradation, whilst the microgrooves on the internal lumen provide a protected environment for ingress of Schwann cells (the supportive cell of the peripheral nervous system), which align on the micro-patterned grooves and aid subsequent nerve regeneration.\n\nExperimental in vitro studies have demonstrated that PCL/PLA blended films are able to support the attachment and growth of Schwann cells21. These cells are key players in nerve regeneration through delivery of neurotrophic factors and extracellular matrix proteins. Furthermore, the microgrooved internal lumen of Polynerve promotes neural regeneration22 and Schwann cell alignment23 to guide the nerve regeneration process. Subsequent in vivo studies on rat sciatic nerve gap of 10 mm demonstrate comparable efficacy of regeneration to nerve graft (current clinical gold standard) in both short (3 week) and long (16 week) timepoints22.\n\nMoreover, in vitro and in vivo evidence demonstrates an improved biological response of the specific micro-grooved architecture of Polynerve when compared to no grooves. Therefore, it is likely to be at a biological advantage when compared to the use of Neurolac® (Polyganics BV) which contains no internal lumen architecture within which to guide the neural regeneration. In randomised clinical trials, Neurolac® is reported to have comparable efficacy to gold standard autograft in defects up to 20mm20. This device is made of similar polymer materials but at a different PCL:PLA ratio. Whilst Polynerve is 4:1, Neurolac® is 1:1 which confers a more rapid degradation and mechanical problems in particular a lack of flexibility. For these reasons, we expect Polynerve to be a superior nerve repair conduit in clinical use.\n\n\nPrimary objective\n\nTo assess the safety and tolerability of use of the polymer biomaterial nerve conduit to repair a sensory nerve transection of the hand.\n\n\nSecondary objective\n\nTo measure efficacy of the polymer biomaterial nerve conduit to support nerve regeneration following transection of the sensory nerve of the hand.\n\n\nProtocol\n\nThis study is a UK-based, prospective, single-centre, unblinded, phase I clinical trial of a novel nerve conduit device. The proposed study will register eligible participants to undergoing repair of a transection of a sensory nerve of the hand using a novel, synthetic nerve conduit polymer. All participants that receive the nerve conduit device will be followed for a period of 12 months post-surgery.\n\nEligible participants will be identified by the Principal Investigator and Co-Investigators within the Department of Burns, Plastics and Reconstructive Surgery at Manchester University NHS Foundation Trust (MFT) outpatient department and trauma database.\n\nParticipants deemed eligible for consideration and potential entry into the study (see Table 1) will be provided with a verbal and written explanation of the study. The participant will be given at least 4 hours and ideally greater than 24 hours to consider participation. The minimum of 4 hours is stated because these are participants with traumatic injuries and will occasionally require or be offered an operation within this time period. After all queries have been addressed and the clinical team is confident that the participant understands the study and all it’s requirements, participants will be consented onto the study.\n\n\nConsent\n\nWritten consent will be taken from potential participants by a member of the trial team who is suitably qualified and experienced, is good clinical practice (GCP)-trained and who has been delegated by the principal investigator to undertake this activity (this delegation will be clearly documented on the delegation log). If a participant is unable to sign the relevant consent form due to physical difficulties resulting from their clinical condition or pre-existing physical condition (e.g. visual difficulties or limb weakness), witnessed consent will be obtained.\n\nDuring the consent process it will be made clear to the participant that they will remain in the trial should capacity be lost unless the decision to withdraw them is made by their representative, the research team, or by their clinical team. A model consent form, alongside the patient information sheet, is available as Extended data24.\n\n\nIntervention\n\nParticipants recruited onto the trial will be operated on by way of routine surgical procedures in an operating theatre at MFT. Local anaesthetic (e.g. 0.5% marcaine and 2% lignocaine ± adrenaline 1:200,000) will be administered as deemed appropriate by the surgeon; and a general anaesthetic will be administered by the anaesthetic team as determined by the clinical situation.\n\nIn standard operating theatre sterile conditions, the wound will be debrided and irrigated as necessary. The digital nerve will be examined under loupe or microscope magnification and a decision made as to the most appropriate surgery method. If the nerve gap is less than 5 mm and the stumps can be co-apted in a tension-free manner, then the nerve will be repaired primarily (end-to-end). If the nerve gap is greater than 20 mm it will be repaired with a nerve graft or as has been decided between the participant and the surgeon. In the instance of a nerve gap between 5 and 20 mm, the Polynerve biomaterial nerve conduit will be used. Three diameters of Polynerve will be available sterilised and packaged: 1.5 mm, 2 mm and 3 mm diameter. All Polynerve conduits are 22 mm in length and will be cut to fit the nerve gap allowing for tension-free epineural suturing of the proximal and distal nerve ends. The nerve will be sutured into the Polynerve conduit with an 9/0 Ethilon suture (Ethicon, UK).\n\nSkin will be repaired with standard treatment of Ethilon suture (Ethicon, UK). The wound will be covered with standard treatment of a barrier dressing such as Mepitel (Mölnlycke Health Care, Sweden), and a secure gauze-based dressing overlying. If concomitant injuries exist such as tendon injuries, then the hand will be dressed using standard treatment including post-operative splinting.\n\nAll participants will receive antibiotics at induction of anaesthetic and for 1 week post-operatively. This will be co-amoxiclav 625 mg three times daily, or if penicillin allergic clarithromycin 500 mg twice daily. This is standard treatment following nerve graft surgery.\n\nStandard post-surgical follow-up will be conducted 1 week and 2 weeks post-surgery, and additional follow up at 3 months, 6 months and 12 months post-surgery will be scheduled (Figure 1 and Table 2).\n\na In the event of repeat surgery, the 3-month, 6-month and 12-month follow-up visits will be performed from the date of the first surgery.\n\nb Sensory outcome assessment to be completed pre-operatively.\n\nThe potential risks to all participants undergoing sensory nerve repair include abnormal scar formation (hypertrophy/keloid), delayed wound healing, pain related to the wound site, neuroma formation, altered sensation of the skin around the wound (paraesthesia), no improvement in recovery of sensation, surgical site infection and bleeding requiring surgery (haematoma). Depending of the severity of these events some may require further surgery.\n\nThe additional potential risks to study participants receiving our polymer nerve conduit are persistent infection, allergy to the polymers PCL/PLA, local irritation and extrusion of the device. Some such risks may require surgical removal of the Polynerve device. These complications have not occurred in any animal studies conducted to date.\n\nAs this was a phase I study, primarily assessing safety of the device, a pragmatic decision on achievable recruitment numbers at a single centre who would be able to complete 12-months follow-up was made, and no formal power calculations were performed. The trial will recruit 17 participants over 12 months at an intended rate of approximately 1 to 2 participants per month. All participants fitting the eligibility criteria will be recruited.\n\nPrimary outcome. The primary outcome is safety following implantation of the Polynerve device. Safety will be assessed based on assessment of the number and degree (based on the Clavien-Dindo classification of surgical complications25) of adverse device effects (ADE) that may occur during the study period26. In order to ensure all ADE are captured, all adverse events (AE) will be assessed and recorded by the P.I. or appropriate co-investigator deputized by the P.I., immediately upon notification to the study team. (A full out-of-hours AE reporting facility is made available to all trial participants following implantation of a study device.)\n\nThe investigator will assess causal relationship between the medical device under investigation and each AE.\n\nAll expected AE/ADEs of grade 3 or above, except infection of the wound any occurrences of grade 2 or above, will be recorded as a serious AE (SAE) or serious ADE (SADE). Appropriate SAE reporting procedures will be followed including sponsor, MHRA and REC reporting—this will be overseen by a Trial Management Group (TMG).\n\nThe following variables will be collected in the case report forms (CRFs) for each ADE:\n\nADE diagnosis/description\n\nThe date of ADE onset and date\n\nClavien-Dindo grade of maximum intensity\n\nWhether the ADE is serious or not (see above)\n\nAssessment of relatedness to device under investigation or other procedure.\n\nOutcome\n\nSafety information and all concomitant medications (that are associated with the surgical procedure) will be reviewed at each follow up visit (Table 2).\n\nAt the end of the study data from initial treatment will be combined in the presentation of safety data. The number of participants experiencing each ADE will be summarised. The number and percentage of participants with ADEs in different categories (e.g. causally related, Clavien–Dindo ≥3 etc) will be summarised by group. SAEs/SADEs will be summarised separately if a sufficient number occur. Any ADE occurring within the defined 12-month follow-up period will be included in the ADE summaries.\n\nSecondary outcome. To measure degree of efficacy of the nerve conduit device, standard sensory outcome measures will be used:\n\ntwo-point discrimination (2PD)27,\n\nthe Weinstein Enhanced Sensory Test (WEST)28,\n\nLocognosia test (adapted methodology from Jerosch-Herold et al. JBJSB 2006)\n\nThese measurements will be assessed at baseline/time of surgery and at 3-, 6- and 12-months after implantation of the device (Table 2). Data will be tabulated and appropriate statistical analysis performed.\n\n\nDemographic data\n\nCharacteristics of the participants, including medical history and disease characteristics at baseline will be listed for each participant and summarised for the whole cohort.\n\nA paper-based data capture system will be used for data collection and query handling.\n\nCompleted CRFs submitted to the clinical trials unit will be entered onto a study database by the trial data manager. The study database is stored on a secure server with controlled access, regular back-up of data and an audit trail of changes made to the data.\n\nDemographic data will be tabulated. All primary outcome data will be described and summarised. Secondary outcome measurement data will be tabulated and mean ± SD of static 2PD, WEST and Locognosia compared to contralateral control nerves will be presented with one-way ANOVA analysis between groups.\n\nTrial participants will have 24-hour access to a plastic surgery registrar and consultant at Manchester University NHS Foundation Trust in case of any AE/ADE. Any treatment or further surgery required will be decided by the C.I. and implemented at the earliest available opportunity. Appropriate safety reporting will be followed by this on-call team.\n\nTMG. A TMG will be established and will include those individuals responsible for the day-to-day management of the trial (including the chief investigator, co-investigators, the trial statistician and the study project manager). The TMG have operational responsibility for the conduct of the trial including monitoring overall progress to ensure the protocol is adhered to and to take appropriate action to safeguard the participants and the quality of the trial.\n\nIndependent data monitoring committee (IDMC). An IDMC will be instituted to review accruing trial data and to assess whether there are any safety issues that should be brought to the participants’ attention, whether any safety amendments should be made or if there are any reasons for the trial should not continue. The IDMC will be independent of the investigators, funder and sponsor.\n\nExpected AEs which may occur as a result of the surgical procedure include:\n\n• Bleeding from the wound site (haematoma)\n\n• Infection of the wound site\n\n• Abnormal scar formation (hypertrophy/keloid)\n\n• Delayed wound healing\n\n• Pain related to the wound site\n\n• Altered sensation of the skin around the surgical site (paraesthesia)\n\n• Development of neuroma\n\nExpected ADEs which may occur as a result of the medical device under investigation are:\n\n• Intractable infection of the surgical site\n\n• Allergy to the polymers PCL/PLA\n\n• Local irritation\n\n• Extrusion of device through the wound\n\nAuthorized representatives of the sponsor, regulatory authority, or an ethics committee may perform audits or inspections at the centre, including source data verification.\n\n\nEthics and dissemination\n\nThe trial will be conducted in accordance with the principles of GCP.\n\nThe South Manchester REC has approved the trial protocol, informed consent forms and other relevant documents e.g. advertisements and GP information letters (REC reference: 17/NW/0111). In addition, the Health Research Authority (HRA) has also given approval for the trial to commence recruitment.\n\nClinical trial authorisation (CTA) has been obtained from the Medicine and Healthcare products Regulatory Agency (MHRA). The protocol and trial conduct will comply with the Medicines for Human Use (Clinical Trials) Regulations 2004 and any relevant amendments.\n\nAny changes in research activity will be reviewed and approved by the Chief Investigator. With the oversight of the sponsor, the subsequent amendment will be categorised as substantial or non-substantial. Any required changes to the CTA or the documents that supported the original application for the CTA and/or ethical approval will be submitted as an amendment to the appropriate ethical and regulatory authorities by the clinical trials unit. Substantial amendments will not be implemented until the REC grants a favourable opinion for the study, confirmation of No Objection is received from MHRA and local R&D department approval.\n\nParticipants will be assigned a unique trial ID via the King's College clinical trials unit randomisation service which will be used throughout their participation in the trial. Any personal data recorded will be regarded as confidential, and any information which would allow individual participants to be identified will not be released into the public domain.\n\nEssential documents are documents that individually and collectively permit evaluation of the conduct of the trial and substantiate the quality of the data collected. Essential documents will be maintained at the Manchester Clinical Trials Unit (CTU), University of Manchester and at the investigator site in a way that will facilitate the management of the trial, audit and inspection. These documents must be retained for a sufficient period of time (at least 15 years) for possible audit or inspection. Documents will be securely stored and access restricted to authorised personnel.\n\nThe main study results will be published in the name of the study, in a peer-reviewed journal, on behalf of all collaborators. The manuscript will be prepared by a writing group, appointed from amongst the TMG.\n\n\nDiscussion\n\nThe proposed intended use of Polynerve is to surgically repair nerve gaps of up to 20 mm. Nerves selected for the Phase 1 clinical trial of this device will be small sensory nerves of the hand. Polynerve (Figure 2) is designed to support the nerve regeneration process by providing a protected environment for ingress of Schwann cells (the supportive cell of the peripheral nervous system), which align on the micro-patterned grooves and support subsequent nerve regeneration. It is designed to be absorbed by the body completely 18 months after implantation by which time nerve regeneration is complete.\n\nThis image demonstrates a standard Polynerve device of 2 mm diameter and 22 mm long (image not to scale).\n\nDevelopment of an improved-quality, faster peripheral nerve repair using a specialised biodegradable nerve conduit has the potential to transform outcomes of peripheral nerve injury.\n\nOur aims are to improve regeneration across a nerve gap, reduce participant morbidity through avoiding the need for a donor nerve grafting operation thereby avoiding donor site scarring and loss of donor nerve function. Of additional benefit will be an improved surgical efficiency in the operating theatre, because the additional nerve graft harvest procedure will not be necessary.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nOpen Science Framework: A phase I trial of a novel synthetic polymer nerve conduit ‘Polynerve’ in participants with sensory digital nerve injury (UMANC). https://doi.org/10.17605/OSF.IO/HG6WB24.\n\nThis project contains the following extended data:\n\nUMANC Informed Consent Form V5 0 13-Aug-2018.doc\n\nUMANC patient information V5 0 13-Aug-2018.doc\n\nOpen Science Framework: SPIRIT checklist for Phase I trial of a novel synthetic polymer nerve conduit ‘Polynerve’ in participants with sensory digital nerve injury (UMANC): Study protocol. https://doi.org/10.17605/OSF.IO/HG6WB24.", "appendix": "Grant information\n\nThis study is funded by the National Institute for Health Research (NIHR) Invention for Innovation (i4i) Programme (Ref: II-LA-0313-20003). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.\n\n\nReferences\n\nGrinsell D, Keating CP: Peripheral nerve reconstruction after injury: a review of clinical and experimental therapies. Biomed Res Int. 2014; 2014: 698256. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKonofaos P, Ver Halen JP: Nerve repair by means of tubulization: past, present, future. J Reconstr Microsurg. 2013; 29(3): 149–64. PubMed Abstract \| Publisher Full Text\n\nRosberg HE, Carlsson KS, Höjgård S, et al.: Injury to the human median and ulnar nerves in the forearm--analysis of costs for treatment and rehabilitation of 69 patients in southern Sweden. J Hand Surg Br. 2005; 30(1): 35–9. PubMed Abstract \| Publisher Full Text\n\nTerzis J, Faibisoff B, Williams B: The nerve gap: suture under tension vs. graft. Plast Reconstr Surg. 1975; 56(2): 166–70. PubMed Abstract\n\nDaly WT, Knight AM, Wang H, et al.: Comparison and characterization of multiple biomaterial conduits for peripheral nerve repair. Biomaterials. 2013; 34(34): 8630–9. PubMed Abstract \| Publisher Full Text\n\nWang S, Cai L: Polymers for fabricating nerve conduits. Int J Polym Sci. Hindawi; 2010; 2010: 1–20. Publisher Full Text\n\nJohnson EO, Soucacos PN: Nerve repair: experimental and clinical evaluation of biodegradable artificial nerve guides. Injury. 2008; 39 Suppl 3: S30–6. PubMed Abstract \| Publisher Full Text\n\nGerth DJ, Tashiro J, Thaller SR: Clinical outcomes for Conduits and Scaffolds in peripheral nerve repair. World J Clin Cases. 2015; 3(2): 141–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGluck T: Ueber Neuroplastik auf dem Wege der Transplantation. Arch für Klin Chir. 1880; 25: 606–16.\n\nKehoe S, Zhang XF, Boyd D: FDA approved guidance conduits and wraps for peripheral nerve injury: a review of materials and efficacy. Injury. 2012; 43(5): 553–72. PubMed Abstract \| Publisher Full Text\n\nJiang X, Lim SH, Mao HQ, et al.: Current applications and future perspectives of artificial nerve conduits. Exp Neurol. 2010; 223(1): 86–101. PubMed Abstract \| Publisher Full Text\n\nNectow AR, Marra KG, Kaplan DL: Biomaterials for the development of peripheral nerve guidance conduits. Tissue Eng Part B Rev. 2012; 18(1): 40–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTian L, Prabhakaran MP, Ramakrishna S: Strategies for regeneration of components of nervous system: scaffolds, cells and biomolecules. Regen Biomater. 2015; 2(1): 31–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArchibald SJ, Krarup C, Shefner J, et al.: A collagen-based nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman primates. J Comp Neurol. 1991; 306(4): 685–96. PubMed Abstract \| Publisher Full Text\n\nLohmeyer JA, Siemers F, Machens HG, et al.: The clinical use of artificial nerve conduits for digital nerve repair: a prospective cohort study and literature review. J Reconstr Microsurg. 2009; 25(1): 55–61. PubMed Abstract \| Publisher Full Text\n\nBushnell BD, McWilliams AD, Whitener GB, et al.: Early clinical experience with collagen nerve tubes in digital nerve repair. J Hand Surg Am. 2008; 33(7): 1081–7. PubMed Abstract \| Publisher Full Text\n\nWangensteen KJ, Kalliainen LK: Collagen tube conduits in peripheral nerve repair: a retrospective analysis. Hand (N Y). 2010; 5(3): 273–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaras JS, Nanavati V, Steelman P: Nerve conduits. J Hand Ther. 2005; 18(2): 191–7. PubMed Abstract \| Publisher Full Text\n\nWeber RA, Breidenbach WC, Brown RE, et al.: A randomized prospective study of polyglycolic acid conduits for digital nerve reconstruction in humans. Plast Reconstr Surg. 2000; 106(5): 1036–45; discussion 1046-8. PubMed Abstract \| Publisher Full Text\n\nBertleff MJ, Meek MF, Nicolai JP: A prospective clinical evaluation of biodegradable neurolac nerve guides for sensory nerve repair in the hand. J Hand Surg Am. 2005; 30(3): 513–8. PubMed Abstract \| Publisher Full Text\n\nSun M, McGowan M, Kingham PJ, et al.: Novel thin-walled nerve conduit with microgrooved surface patterns for enhanced peripheral nerve repair. J Mater Sci Mater Med. 2010; 21(10): 2765–74. PubMed Abstract \| Publisher Full Text\n\nMobasseri A, Faroni A, Minogue BM, et al.: Polymer scaffolds with preferential parallel grooves enhance nerve regeneration. Tissue Eng Part A. 2015; 21(5–6): 1152–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMobasseri SA, Terenghi G, Downes S: Schwann cell interactions with polymer films are affected by groove geometry and film hydrophilicity. Biomed Mater. 2014; 9(5): 055004. PubMed Abstract \| Publisher Full Text\n\nMurphy R, Reid A: A phase I trial of a novel synthetic polymer nerve conduit \"Polynerve\" in participants with sensory digital nerve injury (UMANC). 2019. http://www.doi.org/10.17605/OSF.IO/HG6WB\n\nClavien PA, Barkun J, de Oliveira ML, et al.: The Clavien-Dindo classification of surgical complications: five-year experience. Ann Surg. 2009; 250(2): 187–96. PubMed Abstract \| Publisher Full Text\n\nSafety reporting - Health Research Authority. Reference Source\n\nDellon AL: Sensibility,re-education of sensation in the hand. Baltimore: Williams & Wilkins; 1981.\n\nWeinstein S: Fifty years of somatosensory research: from the Semmes-Weinstein monofilaments to the Weinstein Enhanced Sensory Test. J Hand Ther. 1993; 6(1): 11–22; discussion 50. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56466", "date": "11 Dec 2019", "name": "Dominic M. Power", "expertise": [ "Reviewer Expertise Clinical trials in peripheral nerve repair and regeneration", "conduits and allograft in nerve gap repairDetensioning nerve reapir", "sutureless nerve repair" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol is well-written and explains the trial design and the rationale for the study well.\nIn the introduction the statement that Ref 3 costs of care are estimated at 51K euros needs clarification. This is the cost for a median nerve injury and includes societal costs and loss of production. I think following on from the statement regarding 9000 injuries per annum that this is misleading and requires clarification.\nThe paragraph detailing the option of direct repair should be expanded to discuss the challenges in direct repair of acute nerve injuries. Delay to repair renders the tension greater and the need for debridement may result in unacceptable tension. There are strategies that can be employed to reduce repair site tension and these include using conduits or connectors to bridge the repair site with offloading remote sutures. Papers by Zhu1 and by Neubrech2 detail the efficacy of this approach.\n\"Where the nerve gap exceeds more than 5 mm, there are two fundamental options, either ‘nerve grafting’ or ‘tubulisation’\" I am concerned that this statement may mislead the reader. A gap of 5-6mm may be left in a no gap acute nerve injury with entubulation as a way of detensioning a repair site. The use of conduits to allow bridging of gaps up to 6mm is supported by evidence form Weber and others. Longer gap entubulation is less favourable and beyond 12mm the evidence for existing conduits is poor. The surgeon should consider the options and these are well described in the paper by Ducic et al.3 They include using autograft and also allograft for sensory nerves (limited evidence for mixed nerves).\nI feel that the introduction is too superficial and aims to prepare the reader for a discussion of an improved conduit design but at the cost of an inadequate review of the available options and evidence base. At minimum there should be an analysis of the gap sizes in the Weber paper (poor data interpretation and grouping of larger and medium gaps with small numbers). A recent systematic review on conduits and gap management by DeSlivia et al., 20154 is better to discuss. Particularly as there is recruitment of the 5-20mm gap range a more extensive introductory comment and literature review is warranted.\nThe discussion on the non-absorbable versus bioresorbable conduits is comprehensive.\nThere should be commentary on the sizing of conduits and the issues about transparency assisting use, particularly when there is a use of a conduit in a short gap reconstruction where misalignment can cause problems.\nThere is good explanation of the rationale for the PCL use.\nThere should be clarification of the need for a conduit to allow minimal deformation under loading to prevent disruption of the primitive fibrin clot matrix for axon regeneration. The statement about the mechanical properties of the Polynerve is insufficient and should include a discussion of this aspect of load and deformation.\nIntervention:\nThere is no discussion of nerve debridement.\nThere is no discussion of the number and technique of suture placement to secure the conduit, nor needle type to pass through the conduit. As such the study may not be repeatable by another group.\nIs the antibiotic use for 1 week local policy or is this adapted for this patient population given the use of a synthetic conduit.\nRisks:\nThere is comment on the risk of neuroma in sensory nerve repair. The specific risks of the Polynerve shouldn't include the general risks associated with a bioresorbable conduit as listed but further detail of the risk of nerve displacement from the conduit should be detailed. Extrusion typically refers to ulceration of the conduit through the wound and not to the nerve displacement from the conduit.\nThere is no discussion of the type of US, the technique and findings interpretation as a secondary outcome measure other than listing in the schedule of events. This is not a normal investigation following simple nerve repair or grafting and the rationale and interpretation must be discussed here.\nThe remainder of the monitoring TMG, DMEC, audit and AE reporting is excellent.\nThe conclusion is good but the points raised should be introduced earlier for the reader to understand the concept for this conduit. The failure to review the evidence for allograft is an oversight.\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": "50340", "date": "20 Mar 2020", "name": "Daniel F. Kalbermatten", "expertise": [ "Reviewer Expertise Experimental and clinical trials in peripheral nerve repair and regeneration", "bioarbsorbable nerve conduits in nerve gap repair" ], "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 authored by Murphy et al. is a study protocol for a first-in-human use of a new device called \"Polynerve“ for nerve gap bridging.\n\nIts primary objective is to describe a clinical trial to evaluate the safety and tolerability of use of a new polymer biomaterial nerve conduit to repair a sensory digital nerve gap of the hand. Secodarily, the trial aims at measuring the efficacy of this innovative conduit as an alternative to surgical repair, autologous grafting and other allogenic materials to bridge small gaps of digital nerves.\n\nOverall, the study design is well-structured and the rational of utilizing this new method in a clinical pilot study is clearly explained and outlined.\nThe introduction gives an overview of important impact socio-economic of nerve injury for the affected individual and the societal interest of finding alternative methods to restore continuity in nerve defects and minmize functional and productive loss. Here, reference 3 is quoting a study on the impact of median and ulnar nerve lesions in the forearm, a very severe subset of injury which may yet not be representative for the majority of over 9000 nerve lesions occuring in the UK annually.\nThe authors provide a detailed overview of the requirements of a valuable alternative method avoiding the disadvantages of current treatment methods, such as autologous grafting or commonly used tubulization devices, including non-resorbable and resorbable nerve conduits. In addition, the authors may also mention the modern options of nerve allografting as an emerging method, especially for smaller sized gaps of sensory nerves, e.g. as recently described by Mauch JT et al. (see reference 11).\n\nNevertheless, the discussion clearly explains the rational for using the new material in the planned trial by the specific advantages of Polynerve as a blend of poly-captrolactone (PCL) and poly-lactic-acid (PLA) promoting neural regeneration and Schwann cell alignment to guide the process of axon regeneration by lumen consisting of a special micro-grooved architecture.\n\nThe further paragraphs about the exact design and setting of the study including consent and intervention with a detailed participant timeline and known risks of the procedures are thorough and concise. The methodology regarding data collection and management and statistics are very solid, as well as the description of oversight committees and dissemination.\nThe protocol is completed by a short discussion which summarizes the expected superior qualities of this new biopolymer nerve conduit and the main goals of a promising future study on nerve regeneration.\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": "50341", "date": "10 Jul 2020", "name": "Andrew M. Hart", "expertise": [ "Reviewer Expertise I am a peripheral nerve surgeon in a national service", "with 20 years experience of neurobiological research", "focused around nerve injury and tissue engineering. I edit the world's second largest reconstructive Journal." ], "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 sensible safe approach to the phased development of a novel nerve reconstruction device. It stands in contrast to other products that have been introduced without due testing.\n\nFunctional outcome measurement is challenging, the existing tests being insufficiently specific for fine discriminatory purposes, but the authors suggest a sensible pragmatic approach for this phase of study.\n\nSafety is well addressed.\n\nEthics are well addressed.\n\nData governance is addressed.\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/8-959
https://f1000research.com/articles/8-956/v1	24 Jun 19	{ "type": "Research Article", "title": "Towards a new model for producing evidence-based guidelines: a qualitative study of current approaches and opportunities for innovation among Australian guideline developers", "authors": [ "Steve McDonald", "Julian H. Elliott", "Sally Green", "Tari Turner", "Julian H. Elliott", "Sally Green", "Tari Turner" ], "abstract": "Background: Many organisations in Australia undertake systematic reviews to inform development of evidence-based guidelines or would like to do so. However, the substantial resources required to produce systematic reviews limit the feasibility of evidence-based approaches to guideline development. We are working with Australian guideline developers to design, build and test systems that make creating evidence-based guidelines easier and more efficient. Methods: To understand the evidence needs of guideline developers and to inform the development of potential tools and services, we conducted 16 semi-structured interviews with Australian guideline developers. Developers were involved in different types of guidelines, represented both new and established guideline groups, and had access to widely different levels of resources. Results: All guideline developers recognised the importance of having access to timely evidence to support their processes, but were frequently overwhelmed by the scale of this task. Groups developing new guidelines often underestimated the time, expertise and work involved in completing searching and screening. Many were grappling with the challenge of updating and were keen to explore alternatives to the blanket updating of the full guideline. Horizon-scanning and evidence signalling were seen as providing more pragmatic approaches to updating, although some were wary of challenges posed by receiving evidence on a too-frequent basis. Respondents were aware that new technologies, such as machine learning, offered potentially large time and resource savings. Conclusions: As well as the constant challenge of managing financial constraints, Australian guideline developers seeking to develop clinical guidelines face several critical challenges. These include acquiring appropriate methodological expertise, investing in information technology, coping with the proliferation of research output, feasible publication and dissemination options, and keeping guidance up to date.", "keywords": [ "Clinical guidelines", "Updating guidelines", "Living guidelines", "Qualitative research", "Interviews" ], "content": "Introduction\n\nMany organisations in Australia and internationally undertake and use systematic reviews to inform development of clinical practice guidelines. This process is critical for translating the results of research into evidence-informed decision making and clinical practice to ensure the best possible outcomes for patients1. It is essential that this process is both efficient and cost-effective, particularly given the significant time, effort and resources spent developing guidelines. In Australia, for example, by 2015 around 600 locally developed guidelines were included in the National Health & Medical Research Council (NHMRC) guidelines portal2.\n\nIn 2016, in response to concerns around the trustworthiness of Australian guidelines to inform decision making, NHMRC released a consultation paper ‘Better informed health care through better clinical guidelines’3. The paper identified several challenges facing clinical guidelines in Australia, including concerns over quality, lack of investment in information technology, and obsolescence. These concerns are compounded by the resources required for research synthesis and the sheer volume of research now available.\n\nSystematic reviews are the gold-standard approach for synthesising research evidence and should thus be the foundation of guidelines, but the substantial time and resources required to produce them potentially limit their contribution and uptake into clinical guidelines4. Although this barrier has been partially ameliorated in recent years by the explosion in the number of systematic reviews being published5 (allowing the focus to shift away from undertaking systematic reviews from scratch) guideline developers now have to contend with the problem of keeping up to date with, and making sense of, the burgeoning global research output.\n\nCochrane is a leading international organisation dedicated to ensuring that reliable evidence informs all levels of decision making in health. The routine use of systematic reviews as the evidence base for guidelines has led to numerous collaborations between Cochrane and various guideline groups to facilitate the efficient and more rapid inclusion of Cochrane evidence6,7.\n\nIn a project funded by Cochrane and NHMRC, we are working to develop new tools and systems that will substantially reduce the time and resources required to undertake rigorous systematic reviews8. Our team is working on text mining technologies, machine learning systems and crowd-sourcing to improve the efficiency of study identification, citation screening and data extraction. We are also investigating other tools like automated evidence delivery services that can provide easy access to new, relevant evidence.\n\nIn this study we sought to inform this work by interviewing Australian guideline developers with the aim of understanding their evidence needs and the kinds of tools and services that will enable them to most efficiently access, produce and use reviews of research evidence in guideline development.\n\n\nMethods\n\nWe aimed to understand the evidence needs of guideline developers by exploring how guideline groups manage processes for identifying, appraising and synthesising relevant evidence, and what the implications for these processes are of potential new technologies to support guideline development. We used a qualitative approach (semi-structured interviews) to explore the experiences of guideline developers and to gauge their receptivity to new ways of managing their evidence needs.\n\nEthics approval was provided by Monash University (CF16/1950 – 2016000999). Reporting of the methods and results comply with the consolidated criteria for reporting qualitative studies (COREQ) checklist9. A completed COREQ checklist is available10.\n\nIn 2016, following the release of the consultation paper on better guidelines3, NHMRC held a series of forums for guideline developers to share knowledge and expertise in guideline development. A purposive sample of guideline developers who attended these forums was invited to participate in an interview. The sample captured participants and organisations with diverse levels of expertise and approaches to guideline development. Many of them were familiar with the interviewers and their areas of research as a result of attending earlier NHMRC forums and other similar meetings.\n\nParticipants were emailed invitations to participate in an interview, together with a brief project synopsis and an Explanatory Statement. Participation was voluntary. Acceptance of the invitation to participate in the interviews was taken as evidence of informed consent. No one approached to participate declined to be interviewed. Participants gave their consent to be interviewed on the understanding that data would be reported in de-identified summary form in which no individuals could be identified.\n\nData were collected using a semi-structured interview with guided questions, conducted by telephone or in person. Interview guides are available as Extended data10. The interview schedule and questions were developed by S.M. and T.T. The interviews began with participants describing their experience and involvement with guidelines. The questions then explored several issues: contrasting approaches to guideline development adopted by different guideline groups; specific challenges in developing, publishing and updating guidelines; processes or steps where technical capacity is seen as a barrier; and potential advantages and disadvantages of technology in facilitating more efficient guideline development. Interviewees were encouraged to focus on how their current approach to developing guidelines could be more efficient, and what they saw as the main opportunities and challenges to achieving this.\n\nThe question(s) introducing the main issues were the same for each interview, with follow-up questions tailored according to individual responses. Six interviews (with Melbourne-based guideline developers) took place in person and 10 were conducted by telephone. Most interviews lasted between 30–40 minutes. The first three face-to-face interviews were conducted jointly by S.M. (MA; male) and T.T. (PhD; female), with S.M. conducting the remaining interviews. Both S.M. and T.T. are senior research fellows familiar with guideline development processes, and are experienced systematic reviewers and with extensive experience in qualitative research.\n\nIn addition to taking detailed notes during the interview, interviews were audio-recorded following the consent of the interviewees. Most interviews were one-on-one, but four interviews were with two participants from the same guideline organisation.\n\nNotes taken during the interviews were written up and the audio files transcribed using the transcription service Temi. We used NVivo for Mac 11 to analyse qualitative data and extract quotes. Thematic analysis of the detailed interview notes was undertaken using open coding to identify key concepts that were organised into emerging themes. S.M. undertook the primary data analysis. The initial coding of themes was reviewed by T.T. and together the final conceptual development of themes was agreed. The recordings and transcriptions of the interviews were regularly consulted for clarification and for extraction of quotes. Interviewees were not provided with transcriptions for comment or correction, and no repeat interviews were conducted.\n\n\nResults\n\nWe conducted 16 interviews with Australian guideline developers in August and September 2016. The sample included developers responsible for producing different types of guidelines (e.g. specific versus broad scope), representing both new and established guideline producers with access to widely different resources. The sample was sufficiently large that by the final interviews no new themes were emerging and data saturation was reached.\n\nOrganisations represented included professional colleges and societies (n=3); national bodies for specific diseases or conditions (n=6); specialist, not-for-profit guideline developers (n=1); government-funded guidelines initiatives (n=3); and academic collaborations and partnerships (n=3). Most of these organisations have a long-standing and ongoing involvement in producing guidelines; the participants we interviewed from these organisations tended to have greater technical and process expertise, and thus were able to provide extensive insights into the challenges of managing comprehensive, dedicated guideline efforts. In contrast, the groups involved in developing one-off guidelines had to contend with different concerns, typically relating to funding, time constraints and technical capacity.\n\nAll the organisations interviewed stated a commitment to producing reliable, evidence-based guidance. In practice, this was characterised by organisations that were either actively pursuing endorsement of their guidelines from NHMRC (as a marker of their evidence-based status) or were aware of this option but lacked sufficient time and resources required to pursue approval.\n\nThe two higher order themes identified focused on current challenges and new approaches to guideline production. These themes were further divided into subthemes based around specific challenges and approaches.\n\nThe diversity of organisations and guideline groups represented in the interviews was reflected in the substantial variation in approaches and models described for producing guidelines. However, irrespective of the approach taken or the resources and expertise available, guideline developers faced similar challenges, albeit on differing scales. The key challenges to emerge were capacity restraints around technical and methods expertise; resource constraints around funding and timelines; managing relations with external contractors; dealing with growing volumes of research evidence; publishing arrangements; and updating.\n\nTechnical expertise. Constraints imposed by having limited access to methods expertise, whether internal or external, was a common theme. Guideline groups found it difficult to find suitably skilled staff who could work autonomously; this in turn affected the quantity of synthesis work that could be undertaken at any one time.\n\n“When we were trying to look for casual staff, there didn't seem to be many people around who had experience in running systematic reviews and could help us with a fairly high level of autonomy, and particularly in the diagnostic reviews. It's just a real shortage of skills.” [Participant (P)1]\n\nCompounding limited internal capacity was the under-appreciation by senior staff within the organisation of what is required to develop high quality guidelines.\n\n“Any kind of guide to helping college boards and the like understand what is involved in developing a clinical practice guideline, including resourcing and some idea of cost, would be enormously helpful. I'll just sit at the board meeting and someone will say, ‘We need a guideline on […].’ Well, you know, they have got no concept of what a high quality thing that might be.” [P2]\n\nThe lack of methods expertise and infrastructure to support guideline activities in-house was more keenly felt by smaller guideline groups for whom it was unrealistic to have methodologists on staff, and who relied on the contributions of volunteer guideline group members.\n\nSome guidelines were developed almost entirely on a voluntary basis by clinicians who had little or no input from specialists in evidence synthesis or medical writing, and who lacked the in-depth knowledge of how to derive recommendations using a standardised approach. In one case this led to a series of guidelines from the same organisation all being developed in different ways.\n\n“They were meant to [be done in the same way] but what ended up happening is that because each group was different with a different chair and different makeup … they kind of veered off and did their own thing.” [P3]\n\nEven in circumstances where outsourcing or buying-in expertise was possible, because the guideline work was adequately funded, respondents acknowledged that a degree of internal expertise was needed to communicate requirements and assess the work or service supplied.\n\n“You can definitely have a more effective dialogue with your outsourced reviewers if you have a good feel for the process yourself and if you can use the terminology.” [P4]\n\nResource constraints. A recurring theme was the trade-off between finite resources and rigorous methods. Even for guideline groups able to out-source some activities, the high costs associated with some of these tasks (e.g. conducting systematic reviews to underpin evidence-based guidelines) were often a deterrent. High costs were also perceived to undermine a commitment to rigorous methods, especially when there is a perception that there is little value to be gained. One respondent observed the tension between the pragmatic approach that was acceptable to the guideline group and the gold-standard approach insisted on by the technical specialists, for example by requiring two people to screen all citations and independently extract data.\n\n“They wanted to maintain a certain level of quality from their end, which was fine, but with our guideline reviews, we've tended to do that with one person just because of the resource implications and the need to do things quickly and efficiently.” [P1]\n\nThe perceived value in pursuing rigorous methods versus the opportunity costs in terms of effort and resources led a couple of respondents to question whether the thorough, gold-standard approach to developing guidelines (as is required for NHMRC endorsement of guidelines, for example) delivered a ‘better’ guideline than one developed more pragmatically.\n\n“I'd say the evidence searching and GRADE are the big areas for us, and also that tipping point of how much effort do we put into some processes that aren't necessarily going to change what we currently have as an output. […] I mean, we could go to the nth degree of thoroughness, but I'm not sure that that's going to win us more money or keep us afloat, or meet the needs necessarily of people that use us.” [P5]\n\nRelated to this, the time taken to complete guidelines, and the implications of this for the currency of the guidance, was another common concern, with all steps in the process (searching, screening, synthesis, public consultation) seen as time-consuming. As before, some guideline group members questioned the value of pursuing a rigorous approach.\n\n“We would never publish anything if we did that [follow a rigorous, evidence-based development process.]” [P6]\n\n“As [our chair of the board] says, ‘We never really get it wrong’. So despite the fact that we are not having this absolute thorough, rigorous sort of review of the evidence, nobody ever writes to us and says ‘Oh my God, that’s completely wrong’.” [P6]\n\nUsing external suppliers or contractors. Large guideline groups that had the resources to outsource synthesis work acknowledged the benefits this brought – “the professional help we had … was certainly a major enabling factor” – but also described some challenges. Aside from the costs discussed above, two other issues were mentioned: problems caused when contracted providers changed part-way through, and the difficulties in accessing data and information from contractors.\n\nThe sometimes precarious nature of arrangements with external suppliers was exemplified by stories of work being started by one supplier and completed by another. As well as being a threat to the continuity of the guideline development process, it highlighted the potential lost opportunity to build internal capacity. At the very least, there were implications for workload and opportunity costs associated with wasted effort trying to rectify things. One participant commented:\n\n“You know what it's like picking up somebody else's research work. It's extremely difficult. I was looking in very great detail at the output and I can tell you that was one of the hardest pieces of work that I've ever done because I really had to be absolutely across everything that they’d done.” [P7]\n\nThe second issue, of accessing the backend systems or data generated by contractors so that information can be verified or easily reused, was also problematic.\n\n“A lot [of the work] was done in Excel and Word, and while that’s not completely unreplicable, the problem is all of that data entry – if you want to do it again – you have to do it again.” [P7]\n\nThe implications of limited flows of information and data between guideline developer and contractor may only become apparent later. One participant highlighted the knowledge lost as a result of outsourcing during previous editions of the guideline (e.g. no access to files of original decisions regarding bias assessments), making the process of updating more onerous than it needed to be. Related to this was the use of software and tools to manage specific tasks and produce content (leading to uncertainty over who controls access to the data), and unexpected changes to pricing structures.\n\nInformation challenges. ‘Overwhelmed’, ‘exhausted’ and ‘fatigued’ were just some of the words used to describe members of guideline groups faced with selecting and appraising studies that were potentially relevant to their guideline. In one guideline (that included nearly 300 recommendations) the number of abstracts to screen for the most recent five-year update compared to the previous seven-year period increased by 250 per cent, amounting to over 100,000 abstracts.\n\n“Because it was such an enormous piece of work, there were hundreds and hundreds of questions, and I think what we tried to do was, frankly, just too big for human beings to do.” [P7]\n\n“Well, I mean, all the citation management was done within Reference Manager at the time. There was a lot of Excel. I'm just trying to think back to what we did – it was all very analogue, lots of paper.” [P8]\n\nNone of the respondents had used automation to facilitate citation screening or study appraisal but could see the potential for efficiency gains and were keen to learn more.\n\n“In a perfect world we’d get a machine to look at those abstracts… These days the technology does exist – we should be exploring it – and that would have saved months of human endeavour. It mightn’t be perfect but it’s probably as perfect as a human.” [P9]\n\nOne respondent commented on the twin challenge of having an abundance of evidence for some questions and yet limited evidence for others, making the task of deriving recommendations equally challenging whichever the scenario.\n\nPublishing and dissemination arrangements. Publishing options for guidelines are constantly evolving and cover both traditional print and newer digital media. Despite innovations in publishing, several respondents perceived printed guidelines to be the preferred format of their users; this was especially so for guidelines that serve rural and remote practitioners, who are reluctant to switch to digital-only when reliable online access can’t be guaranteed. This potentially restricts the kinds of publishing and dissemination options available to guideline groups, even if the technical capacity and desire exists to do so.\n\n“We get a lot of people saying ‘I'm doing a remote placement and I've got your books but they're too heavy to carry’ – they’re around five kilos and if you're flying in a small plane, that's half your luggage quota gone. The challenge for us is we target remote areas – internet access isn't necessarily good and so a website isn't necessarily useful.” [P10]\n\nWhile two of the largest groups, responsible for comprehensive, all-of-condition guidelines, only publish online, this is the exception. Most developers provide an online version of their printed guidelines in PDF format – “a thick PDF you’ve got to wade through” – but that is often the extent of the digital functionality.\n\n“We had a big effort to make our website mobile friendly so it looks ok, but the ultimate thing is that we are still down to PDFs. It’s really very tricky.” [P5]\n\nThe challenges some guideline groups face in going beyond traditional dissemination formats include constraints due to existing technology platforms (one group was still running Windows 7); variations in connectivity across jurisdictions (metropolitan versus rural); limited capacity to influence technology decisions (e.g. government decisions); and few resources to spend on publishing after the synthesis effort. Nevertheless, there is a broad willingness to embrace more responsive and digital publishing models.\n\n“We'll be really pushing the electronic. …We want to make it more electronic and more responsive.” [P8]\n\n“We do want to move more online … to be more responsive.” [P11]\n\nIt was also acknowledged that any new publishing formats are likely to be in addition to what is currently offered, further stretching constrained resources. For groups developing guidance that is more targeted at patients and carers, social media is another form of dissemination being considered.\n\n“We're also looking at more innovative ways of disseminating the content for consumers and carers as well... There's an incredibly huge role for social media and electronic media that we can use to engage consumers rather than the old fashioned printouts.” [P8]\n\nUpdating. Of all the challenges with the current process of guideline development, updating guidelines generated the most discussion. For some respondents, updating was a looming challenge that was either not yet an issue or had been deliberately put to one side until after the completion of the current edition of the guidelines.\n\n“But with these [guidelines], because it was short-term funding, we didn't really think that much ahead.” [P12]\n\nMany reflected on the realisation that updating should be a key consideration in the planning and conduct of any guideline. One respondent suggested that updating should be the first item on the agenda for any guideline group about to embark on the development of a new guideline, not least because of the implications for publishing arrangements.\n\n“Updating would have driven decisions about whether [the guidelines] were produced electronically or in print.” [P7]\n\n“We were very diligent about thinking of uptake and implementation right at the outset … but what wasn’t front and centre of our minds was how the hell were we going to update these monsters once they were developed.” [P7]\n\nFor the more-established guideline groups – those who have been through one or more cycles of guideline development and revision – how best to approach updating was an increasing focus of their guideline management. Conscious of the expense, time and personnel involved in producing updated editions of guidelines, respondents were exploring or implementing alternatives to conventional updates. However, as one respondent noted, willingness to move in this direction is not sufficient without the rest of the guideline apparatus being in place.\n\n“A restriction of working within [named organisation] is the technology is not there. We don’t have the funding for anything innovative, even to trial. Is it worth investing in regular updates if we can do nothing or we don’t have the capacity to incorporate them?” [P5]\n\nIn considering what the future holds for guideline production, respondents focused discussion around improving efficiencies, accessing appropriate expertise and finding alternative approaches to updating. Several respondents recognised that adopting a more flexible model of updating guidance was not only desirable but also essential for ensuring sustainability of the whole guideline effort.\n\nEfficiencies in searching, screening and data extraction. Many respondents had attended meetings at which several new tools and technologies to support evidence synthesis had been presented and discussed, and were thus familiar with the potential of these technologies to accelerate guideline development processes, particularly around the time-intensive aspects, such as screening and data extraction. For scenarios where eligibility criteria around study design are clear (e.g. focus on systematic reviews and randomised trials), several respondents were aware of tools that classify citations according to the probability of being relevant or irrelevant, and of the potential time savings they offered.\n\n“If there were ways of text mining abstracts to be able to get a much quicker decision – ‘Is this abstract going to be relevant?’ – I think that would be wonderful.” [P13]\n\nNone of the respondents had yet used these technologies, such as machine learning or crowd-sourcing, so while there was enthusiasm for experimenting to minimise screening workload, acceptability was an issue. Reservations were raised about what might be missed by relying on automation, as well as the loss of immersion in a topic that comes from iteratively screening the literature.\n\n“My concern … is that when you exclude a study, it just goes into an excluded studies bucket and you can’t really get it back out.” [P8]\n\n“I think it's really important you get people doing the screening who really understand the context of the guideline. […] For intervention topics it can be a lot more straightforward but, like I say, for psychosocial screening questions and risk factor assessment type questions, it's much more nuanced.” [P8]\n\n“If I was crowd-sourcing the screening I would be very concerned about the potential for excluding studies that should be included.” [P8]\n\nLike screening, the tasks of extracting data and assessing the validity of studies can be very resource-intensive. Several respondents were aware of systems and tools that could automate risk of bias assessments and aspects of data extraction but were yet to use them.\n\n“It would be very interesting to see if that [automation] could help with data extraction, etc. … which is time-consuming and quite taxing … reading hundreds of papers, it’s quite difficult at times. I think it would make a huge difference to our time and our resources.” [P14]\n\nSynthesis and GRADE expertise. There were several steps in the evidence synthesis process where respondents felt they could benefit from help. Many respondents emphasised their lack of research synthesis expertise, whether among in-house staff or among guideline working group members. This issue varied depending on whether groups saw their primary role as translating evidence to produce practical guidance or doing the evidence synthesis. Those guideline groups that conduct their own evidence syntheses would like to access external specialists to conduct these in a timely and efficient way.\n\n“There will be occasions when we will need to do our reviews from scratch. I think that's where we would like to have support where we can.” [P11]\n\nMany commented on the rapid evolution of methods to assess the certainty of evidence and derive recommendations in guidelines, and were grappling with the transition from previous rating systems, such as the NHMRC’s FORM, to the more commonly used GRADE that NHMRC has now adopted as its preferred system when endorsing guidelines. The use of GRADE for deriving recommendations was cited as one area where guideline members do not have the time to undergo training and there is a lack of internal capacity to deliver it.\n\n“If we can access software or any kind of expert organisation that can help us to do systematic reviews, and as part of the GRADE process, that would be extremely helpful for us. That's currently a pretty intensive process and we don't have the resources to do it in house.” [P11]\n\n“We'd like to educate our members as we move to GRADE. We'd like them to know what are the different terminologies that are used and get them to understand the differences in how you GRADE evidence and how you read the evidence. … So that when they start seeing these recommendations they fully understand why we say moderate evidence rather than the existing 1B or 2B.” [P12]\n\nFor more experienced guideline developers, the transition to GRADE and using the software to produce the evidence tables was less of an obstacle. One respondent when asked if they felt adequately supported in how to use GRADE said:\n\n“I think so. I mean we just read the papers, looked at GRADEPro, watched some webinars and that was enough. I don't know what more benefit would have been to have had face-to-face training. They were quite good with email fortunately; if we had any questions and we emailed them, someone would always get back to us fairly quickly.” [P1]\n\nSmarter use of existing evidence and closer Cochrane links. In the face of funding and time constraints, the necessity of pragmatic approaches to guideline development was frequently raised by participants. The abundance of guidelines produced globally gives groups more options when considering adapting existing high-quality evidence-based guidelines. Given the high costs of conducting a systematic review relative to the resources available for the guideline as a whole, guideline groups were understandably keen to identify cost-efficient, pragmatic approaches that achieved good outcomes without compromising quality or reliability.\n\n“We just don't have the resources to undertake systematic reviews … so we adopt a much more pragmatic approach to how we develop our guidelines.” [P12]\n\n“There are times when we will develop recommendations from scratch, but that is quite rare and that's usually when we have grants and a lot of resources to support us.” [P11]\n\nThe proliferation of published systematic reviews in recent years increases the likelihood that reviews already exist that fully or partially address questions in guidelines.\n\n“There's been an enormous volume of evidence published since the last guideline and so a lot of it has just been about having a really sensible approach to the update, and very much with a view to finding existing systematic reviews.” [P1]\n\nAs groups look to adopt efficiencies in how their guidelines are developed and kept up to date, there was a clear role for Cochrane reviews to inform recommendations. However, while one respondent had contracted Cochrane to update a review, in general there was limited contact with Cochrane groups to help prioritise reviews for updating.\n\n“If there's an existing Cochrane review or an existing guideline that's used Cochrane methods – the NICE guidelines meet that criterion – then we use those as our foundation reviews. Just update those.” [P8]\n\nOne guideline developer commented on their positive experience with using Cochrane reviews when they were reasonably up to date, but also on their frustration when they were several years old or only partially reported outcomes relevant to the guideline. This necessitated updating searches, going back to the primary studies and doing GRADE assessments, so that it was sometimes more efficient to ignore the existing Cochrane review and start again.\n\n“It would be great if you have found your Cochrane review and it's three years out of date and you'd like it updated, that would be fantastic. I guess the challenges are ones around timing and […] can it fall within the overall timeframe of the guideline. The other issue is around scope and […] what studies are included or excluded and what happens when the guideline group doesn't agree with the Cochrane review group.” [P8]\n\nOne guideline group, that had drawn extensively on over 75 Cochrane reviews to inform their recommendations, felt that closer links with the respective Cochrane group (e.g. to align with priorities around updates or new reviews), as well as other guideline groups internationally, would be a much smarter way of collaborating.\n\nAnother guideline developer that makes use of existing systematic reviews was cautious about the reliability and variable quality of many so-called systematic reviews now being published.\n\n“Need to be sceptical about the methods others have used to do their systematic reviews. Some are not good quality.” [P15]\n\nNew approaches to updating. Several ideas were raised in relation to alternatives to the one-off guideline update. They included implementing an evidence notification system for hot topics, rather than relying on ad hoc approaches involving content experts, even if these specialists typically know if papers have been published that will impact on existing questions and recommendations. Similarly, an automated signalling service or horizon-scanning process would be a systematic way of identifying newly available research.\n\n“The idea of more automatically checking what's out there in the literature and bringing the results back would be very helpful, and may also to some extent resolve my concerns about the wrong sort of human influence [selectively picking their favourite studies].” [P4]\n\nSince the strength of the evidence underpinning recommendations varies, being able to ‘switch off’ questions because the evidence is highly stable and instead focus on those questions where there is uncertainty, was proffered as an efficient approach to adopt but, in contrast to the initial guideline development process, the guidance around updating was less clear.\n\n“I think we're really a little bit ad hoc, we don't have any kind of notification system.” [P14]\n\nThe absence of a formal or consistent process for determining where to focus updating efforts was evident across the guideline groups. Some groups had already moved towards having dedicated updating staff (screening records, identifying new questions) and could see the benefits in using PICO-based tools (that use machine learning to determine the relevance of publications for particular topics) to streamline this process.\n\nA cautionary note was struck by some respondents who expressed mixed feelings toward adopting a continual updating process. One concern was the impact on users of too-frequent changes to guidance, as well as how guideline groups would manage communication around these changes. Another was the risk of selectively identifying studies on a prospective basis instead of conducting comprehensive searches. Such a process would need to be transparent and require the dispassionate application of pre-specified criteria.\n\n“I have mixed feelings about the continuous update process, I think its strength is that the guidelines aren't getting out of date by the time the five-year, or whatever interval is chosen, comes up. On the other hand, there is a danger with the continuous update process that it gets triggered when somebody's favourite paper gets published… The process is meant to be more dispassionate, more arms length than that.” [P4]\n\nSeveral guideline developers expressed wariness in going back to their content experts with requests for ongoing involvement when those experts were already ‘extraordinarily fatigued’ by the push to produce the most recent update to the guideline. The resources and goodwill required to sustain advisory groups in a more responsive ‘living guidelines’ model have yet to be explored.\n\n\nDiscussion\n\nClinical practice guidelines are an essential part of modern health care, helping to translate research into practice, inform policy decisions and improve patient outcomes. Their existence across all areas of health demonstrates their importance in supporting clinicians, patients and managers in making health-related choices. However, substantial resources and organisational commitment are required to produce and maintain guidelines. The Institute of Medicine contends that trustworthy guidelines should be informed by a systematic review of the evidence, follow explicit and transparent processes to minimise bias, and provide ratings of both the certainty and strength of recommendations1. Guidelines should also be revised “when important new evidence warrants modifications of recommendations”1. Even without the burden of keeping recommendations up to date, producing guidelines that meet accepted standards is a resource-intensive endeavour, requiring technical expertise, significant institutional support and the abundant commitment of guideline panel members.\n\nIn this study we explored the experiences of a diverse sample of Australian guideline developers and sought to understand their views regarding new ways of managing their guideline evidence needs. Through semi-structured interviews, we identified several challenges: limited technical expertise; constrained funding and timelines; interactions with external contractors; addressing information challenges; modernising publishing arrangements; and updating guidelines. Our findings reflect and expand upon the challenges facing clinical guidelines in Australia that were identified in a discussion paper from Australia’s NHMRC3. Three of these link directly to what we found: lack of capacity (methodological expertise); lack of investment in information technology (widespread existence of manual systems, and paper or pdf publications); and obsolescence (keeping guideline recommendations up to date). This last challenge was seen as critical for several guideline developers we interviewed.\n\nSystematic reviews that rigorously identify, appraise and synthesise relevant evidence to answer clinical questions are fundamental to evidence-based guidelines. Producing these systematic reviews requires guidelines developers to be, or to have access to and funding to support, “skilled groups of professionals with expertise in the methodology of evidence synthesis, experience in the application of these methods and the design, conduct and interpretation of systematic reviews”3. Our findings concur with those of the NHMRC who identified that Australia lacks “the necessary critical mass of expertise and experience in the design and conduct of systematic reviews”, and that this is a crucial barrier to evidence-based guideline development. Our participants noted that a range of strategies are needed to overcome this challenge, including training to build workforce capability (e.g. GRADE Centres to provide support in key methods), establishment of consistent standards and approaches that are widely adopted, and which facilitate development of skills that are transferable across guideline projects (e.g. NHMRC guidelines for guidelines)11 and exploring effective methods of international collaboration to enable reuse of evidence syntheses.\n\nThe sustainability and updating of evidence-based guidelines may be enabled by various technological developments that can maximise efficiency, especially for tasks like searching and screening that are most amenable to automation. Within Cochrane, for example, combining machine learning with crowd-sourcing has resulted in a high-performing study classifier for identifying randomised trials being implemented in review workflows and facilitating living systematic reviews12. The guideline developers interviewed for this study were aware that these kinds of new technologies were becoming more routine and were keen to see how they could be used to save time and resources. However, as a recent survey among systematic reviewers has shown13, uptake of these tools is impeded by several barriers, including mismatch to workflows, licensing, steep learning curve and lack of support.\n\nPart of the rationale for developing systems that can handle more flexible approaches to updating is the phenomenal growth in global research output14. Research synthesis, a discipline that evolved partly in response to the increase in primary research, is itself susceptible to the same trends. For example, around 25,000 systematic reviews are added to the Epistemonikos database annually and monthly registrations of systematic reviews in PROSPERO, the international prospective register of SRs, have increased 10-fold over the past five years15. The challenge for guideline developers is clear: how can guidance feasibly be kept up to date when faced with this continual onslaught of evidence.\n\nNot surprisingly, the three-to-five-yearly comprehensive guideline update is increasingly seen as impractical, and in some cases has already been abandoned in favour of more ‘living’ approaches, acknowledging that feasibility and sustainability are key issues. As a consequence of the various process constraints described in this paper, there is a strong desire for a guideline development model that explicitly recognises current limitations and invests in smarter, more efficient ways of maintaining and updating guidance.\n\nAlthough the concept of living guidelines is not new, their uptake has been limited by practical considerations. A survey in 2009 of 40 guideline developers showed that while a majority of institutions supported the concept of living guidelines, they were regarded as very labour-intensive and requiring extra resources16. In a 2014 review of guidance for updating provided in methodological handbooks for guideline developers, Vernooij found “the majority do not provide guidance for the literature search, evidence selection, assessment, synthesis, and external review of the updating process”17. In the same year, in a systematic review of methods for updating clinical practice guidelines, partial updating was deemed more appropriate because it could be tailored to the different needs of the topic (i.e. dynamic versus stable evidence base)18. The review concluded that guideline developers should “implement a systematic updating procedure that includes an ongoing monitoring system”.\n\nNew, more dynamic approaches to updating guidelines would require the development of systems and tools to manage the flow and identification of potentially relevant new evidence, prioritise recommendations for surveillance, as well as establish new processes for managing the expert working groups involved in reviewing the evidence and revising the recommendations.\n\nWe applied purposive sampling to ensure we captured the perspectives and experiences of a variety of guideline developers. Groups represented in our interviews ranged from organisations responsible for substantial national programs of guideline activity through to smaller, ad hoc guideline groups convened to cover more narrowly-focused topics. The themes that emerged from the interviews, especially around issues affecting the production and updating of guidelines, were often common to all guideline groups regardless of size, longevity or funding. The in-depth and semi-structured nature of the interviews allowed for a richer understanding of the evidence needs facing guideline groups than could have been achieved through surveys or questionnaires. Although the inclusion of Australian-only guideline producers may limit the applicability of the findings, our informal interactions with guideline groups internationally often highlight similar themes, suggesting these findings are generalisable to other settings.\n\nTrustworthy clinical practice guidelines are an important knowledge translation tool but the challenge of producing and then keeping these guidelines up to date has been a perennial one for guideline developers. The concept of a ‘living’ guideline in which new evidence is incorporated as it is identified is one that is gaining momentum19. This has partly been enabled by the growing popularity of online platforms to manage the conduct of evidence syntheses, as well as innovations in linked data and machine learning. For example, machine learning has several applications for guidelines, including the development of classifiers to automate the retrieval of relevant research and to model the risk of conclusions of systematic reviews changing20.\n\nCollaborations between guideline groups internationally may also increase the feasibility of maintaining living guidelines. In a partnership between the Stroke Foundation (Australia) and Cochrane Australia, we are aiming to support the translation of health research into practice by piloting living guidelines for stroke management7. A similar project is underway in diabetes where four organisations have come together to pilot living recommendations in two priority areas of diabetes care21. As well as evaluating the feasibility of processes such as continual evidence surveillance and rapid synthesis updates, both these initiatives address the challenges of obsolescence and lack of investment in information technology that were highlighted in the NHMRC report on the state of clinical guidelines in Australia3. Future research should also evaluate the relative burden (costs, time, effort) of more continual approaches to updating compared to the stop-start nature of intermittent updating.\n\n\nConclusions\n\nOur research has defined the key challenges and needs faced by Australian guideline groups in developing rigorous evidence-based guidelines. Aside from the constant challenge of managing financial constraints, we identified several critical needs, including acquiring appropriate methodological expertise; investing in information technology; coping with the proliferation of research output; feasible publication and dissemination options; and keeping guidance up to date. For the guideline effort to be sustained, technological innovations (around platforms, tools, services and data) need to drive the efficiency of guideline development processes so that guideline developers can adopt more flexible, sustainable and still rigorous approaches to updating guidance.\n\n\nData availability\n\nGuideline developers agreed to participate in the study on the basis of individual and organisational anonymity. The content of the audio recordings and transcripts contain information that would identify individuals and organisations. As a consequence, access to the audio-recordings and transcripts of the participant interviews, together with the content analyses, is unavailable.\n\nOpen Science Framework: Australian guideline developers: needs analysis. https://doi.org/10.17605/OSF.IO/H56TF10.\n\nThis project contains the following extended data:\n\nMcDonald_AusGuidelines_interview_guide.docx (interview guide used in this study).\n\nOpen Science Framework: COREQ checklist for “Towards a new model for producing evidence-based guidelines: a qualitative study of current approaches and opportunities for innovation among Australian guideline developers”. https://doi.org/10.17605/OSF.IO/H56TF10.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\n\n\nS.M., S.G., J.E. and T.T. conceived the idea of the study. S.M. and T.T. developed the interview questions and conducted the interviews. S.M. transcribed the data and performed the initial coding of the data. S.M. and T.T. refined the thematic analysis and drafted the manuscript. All authors revised the manuscript critically for important intellectual content and approved the final version submitted for publication.\n\n\nGrant information\n\nThis study is supported by funding from Cochrane and the Australian National Health & Medical Research Council (Partnership Project grant APP1114605).\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\nThe authors thank the guideline developers for their participation and willingness to be interviewed, and NHMRC for facilitating contact with the guideline groups.\n\n\nReferences\n\nInstitute of Medicine (US) Committee on Standards for Developing Trustworthy Clinical Practice Guidelines, Graham R, Mancher M, et al.: Clinical Practice Guidelines We Can Trust. Washington DC: National Academic Press; 2011. PubMed Abstract \| Publisher Full Text\n\nNational Health and Medical Research Council: Australian Clinical Practice Guidelines. Reference Source\n\nNHMRC: Better informed health care through better clinical guidelines: an NHMRC Draft Discussion Paper. 2015. Reference Source\n\nGhersi D, Anderson WP: Can Australia's clinical practice guidelines be trusted? Med J Aust. 2015; 202(1): 8. PubMed Abstract \| Publisher Full Text\n\nPage MJ, Moher D: Mass Production of Systematic Reviews and Meta-analyses: An Exercise in Mega-silliness? Milbank Q. 2016; 94(3): 515–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTovey D: The role of The Cochrane Collaboration in support of the WHO Nutrition Guidelines. Adv Nutr. 2014; 5(1): 35–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnglish C, Bayley M, Hill K, et al.: Bringing stroke clinical guidelines to life. Int J Stroke. 2019; 14(4): 337–339. PubMed Abstract \| Publisher Full Text\n\nProject Transform. 2019. Reference Source\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Heatlh Care. 2007; 19(6): 349–57. PubMed Abstract \| Publisher Full Text\n\nMcDonald S: Australian guideline developers: needs analysis. 2019. http://www.doi.org/10.17605/OSF.IO/H56TF\n\nNHMRC: Guidelines for guidelines. 2019. Reference Source\n\nThomas J, Noel-Storr A, Marshall I, et al.: Living systematic reviews: 2. Combining human and machine effort. J Clin Epidemiol. 2017; 91: 31–7. PubMed Abstract \| Publisher Full Text\n\nvan Altena AJ, Spijker R, Olabarriaga SD: Usage of automation tools in systematic reviews. Res Synth Methods. 2019; 10(1): 72–82. PubMed Abstract \| Publisher Full Text\n\nBornmann L, Mutz R: Growth rates of modern science: a bibliometric analysis based on the number of publications and cited references. J Assoc Inform Sci Technol. 2015; 66(11): 2215–22. Publisher Full Text\n\nPage MJ, Shamseer L, Tricco AC: Registration of systematic reviews in PROSPERO: 30,000 records and counting. Syst Rev. 2018; 7(1): 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlonso-Coello P, Martínez García L, Carrasco JM, et al.: The updating of clinical practice guidelines: insights from an international survey. Implement Sci. 2011; 6(1): 107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVernooij RW, Sanabria AJ, Solà I, et al.: Guidance for updating clinical practice guidelines: a systematic review of methodological handbooks. Implement Sci. 2014; 9: 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker M, Neugebauer EA, Eikermann M: Partial updating of clinical practice guidelines often makes more sense than full updating: a systematic review on methods and the development of an updating procedure. J Clin Epidemiol. 2014; 67(1): 33–45. PubMed Abstract \| Publisher Full Text\n\nAkl EA, Meerpohl JJ, Elliott J, et al.: Living systematic reviews: 4. Living guideline recommendations. J Clin Epidemiol. 2017; 91: 47–53. PubMed Abstract \| Publisher Full Text\n\nBashir R, Surian D, Dunn AG: The risk of conclusion change in systematic review updates can be estimated by learning from a database of published examples. J Clin Epidemiol. 2019; 110: 42–49. PubMed Abstract \| Publisher Full Text\n\nCochrane Australia: Australian Living Evidence Consortium. Reference Source" }	[ { "id": "50339", "date": "27 Aug 2019", "name": "Tracy Merlin", "expertise": [ "Reviewer Expertise Clinical practice guideline development and methodology", "evidence synthesis", "systematic reviews and meta-analyses", "health technology assessment" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is a well written and thoughtful analysis of guideline developers' views on the challenges associated with guideline development in Australia. Many of the issues that were raised resonated with my experience of guideline development in Australia.\nI note two points that may have limited the applicability of the conclusions that have been drawn.\nThe study was jointly funded by Cochrane Australia and the NHMRC and the sampling strategy was purposive and drawn from attendees at NHMRC guideline development forums. It is therefor possible that the views that have been ascertained primarily relate to developers' experiences with developing NHMRC guidelines, as opposed to other guidelines that do not have NHMRC endorsement. I am aware of several guideline development groups that choose not to receive NHMRC endorsement in Australia because they find the guideline development requirements of the NHMRC overly restrictive. It is possible that these guideline developers would have had different perspectives on these challenges.\n\nIt would have been helpful to know the degree of experience in guideline development of the different study participants (i.e. the number of guidelines completed) and whether their views varied as a consequence. This was touched on when discussing the theme of \"Synthesis and GRADE expertise\". I would have liked to know whether it emerged with any other themes. This would help with understanding whether the themes identified are generalisable to all types of guideline developer. However, I do note that there was variation in size, longevity and funding of the different guideline groups interviewed.\n\nThe paper has concentrated on machine learning, crowd sourcing and automation as being possible solutions to the need to maintain high standards (good quality systematic reviews) but done in a way that would be time and cost-efficient. It was clear from the Results that although these methods are appealing in concept, very few of these methods have been trialled by guideline groups. It might be useful to reflect on this in the Discussion. What sort of time and cost efficiencies have been empirically found - to date (as I know the field is moving rapidly) - with these sorts of solutions?\nThere was a short section on pragmatic review solutions in the Results section but this was not picked up in the Discussion. Was there any discussion of rapid review methodologies by the study participants, and how these are being used? Or use of overviews of systematic reviews? Would the authors like to comment on the reliability of a well done rapid review versus a comprehensive systematic review, or discuss the empirical evidence reporting on the merits of these? Rather than adopting new technologies and IT to solve the labour and cost-problem, is the solution being more pragmatic about the evidence-base i.e. what is the additional yield from doing a systematic review that has canvassed numerous databases, and conducts independent duplicate screening, extraction and/or critical appraisal, over other types of systematic review?\nSome of the points raised concerning external suppliers seemed to fit better with the updating theme.\nParticipants 7 and 8 seemed to have more quotes used in the paper than others. Were their points supported by other participants in the study?\nThe points raised regarding living reviews were interesting, particularly regarding the need to have predefined criteria for doing proactive horizon scanning or updated searching, rather than triggering an update due to publication of a known new paper. There is less room for selection bias if the process is standardised.\nIt was encouraging to see this sort of research being done. The authors are to be congratulated on reflecting on these issues and considering how evidence based guideline methodology may be improved upon.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "53163", "date": "10 Sep 2019", "name": "Amanda Leach", "expertise": [ "Reviewer Expertise Aboriginal and Torres Strait Islander 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\nPlease note that I am not a qualitative researcher so I have not made comment on these methodological matters. This report on the experience of 16 Australian Guideline developers in Australia is a very useful reality check for those embarking on, or responsible for updating, Clinical Practice Guidelines. I agree with reviewer Martin that the purposive sampling from “those who attended NHMRC-hosted forums” is likely to introduce bias. The authors could provide details of how many developers attended these forums, and potential bias due to familiarity of interviewees with interviewers. It would be of interest to interview developers who were not seeking NHMRC endorsement. The article covers the challenges of data extraction and synthesis well, exploring participant views on potential solutions including technologies such as machine learning. Whilst these are designed to improve efficiency, potential compromises to reliability of Guidelines should be highlighted as an area for future research. I would have liked to have learned more about efficient methods for disseminating guidelines or updates and monitoring the uptake and use of new Guidelines and updates, as well as for opportunities for users to score utility of the Guideline. There is potential for data to be collected on citations, number of digital downloads, the density of hits could identify hot and cold spots where and when Guidelines are being utilised and where and when they are not. Leading to targeted efforts to improve uptake in ‘cold spots’. Updating was also very interesting and important – targeting searches towards clinical queries where there is low confidence and having questions that could be ‘switched off’ is a pragmatic option. One of the issues with updating is how to handle assessing the impact of updates on Guideline recommendations – keeping a review group over time who can determine whether an update changes current recommendations or changes the level of certainty or confidence in a recommendation, and what should be ‘published’. This is perhaps briefly touched on by “… without the rest of the guideline apparatus being in place.” When the evidence is low quality, new data could have a significant impact on a recommendation. In this instance there is also potential for recommendations to ‘flip-flop’ over time, leading to frustration and perhaps distrust of the Guideline. The article raises critical and urgent need for Australia to invest in research to answer the many challenges set out by the respondents. I don’t have specific recommendations for change, and look forward to reading more on progress in this space. Without such progress there will surely be a threat to quality of health care in Australia.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-956
https://f1000research.com/articles/8-954/v1	24 Jun 19	{ "type": "Research Article", "title": "Risks and seasonal pattern for mortality among hospitalized infants in a conflict-affected area of Pakistan, 2013-2016. A retrospective chart review.", "authors": [ "Babette van Deursen", "Annick Lenglet", "Cono Ariti", "Barkat Hussain", "Jaap Karsten", "Harriet Roggeveen", "Debbie Price", "Jena Fernhout", "Ahmed Abdi", "Antonio Isidro Carrion Martin", "Annick Lenglet", "Cono Ariti", "Barkat Hussain", "Jaap Karsten", "Harriet Roggeveen", "Debbie Price", "Jena Fernhout", "Ahmed Abdi", "Antonio Isidro Carrion Martin" ], "abstract": "Background: In recent years, Médecins Sans Frontières has observed high mortality rates among hospitalized infants in Pakistan. We describe the clinical characteristics of the infants admitted between 2013 and 2016 in order to acquire a better understanding on the risk factors for mortality. Methods: We analyzed routinely collected medical data from infants (<7 months) admitted in Chaman and Dera Murad Jamali (DMJ) hospitals. The association between clinical characteristics and mortality was estimated using Poisson regression. Results: Between 2013 and 2016, 5,214 children were admitted (male/female ratio: 1.60) and 1,178 (23%) died. Days since admission was associated with a higher risk of mortality and decreased with each extra day of admission after seven days. The first 48 hours of admission was strongly associated with a higher risk of mortality. A primary diagnosis of tetanus, necrotizing enterocolitis, prematurity, sepsis and hypoxic-ischemic encephalopathy were strongly associated with higher rates of mortality. We observed an annual peak in the mortality rate in September. Conclusions: The first days of admission are critical for infant survival. Furthermore, the found male/female ratio was exceedingly higher than the national ratio of Pakistan. The observed seasonality in mortality rate by week has not been previously reported. It is fully recommended to do further in-depth research on male/female ratio differences and the reasons behind the annual peaks in mortality rate by week.", "keywords": [ "Infant mortality", "Epidemiology", "Seasonal pattern", "Pakistan", "MSF" ], "content": "List of abbreviations:\n\n95%CI 95% Confidence Intervals\n\naRR Adjusted Rate Ratio\n\nDHS Demographic and Health Survey\n\nDMJ Dera Murad Jamali\n\nHIE Hypoxic-ischemic Encephalopathy\n\nMoH Ministry of Health\n\nMSF Médecins Sans Frontières\n\nMSF-OCA Médecins Sans Frontières Operational Centre Amsterdam\n\nNEC Necrotizing Enterocolitis\n\nRR Rate Ratio\n\n\nIntroduction\n\nAs part of the Millennium Development Goals, the under-five mortality (U5M) should be reduced by two-thirds from 1990 to 20151,2. In 2015, the U5M was reportedly 47 per 1,000 live births in low resource settings compared to 6 per 1,000 live births in high resource countries2. Mortality in children under five is still high in Southern Asia compared to other regions. In Pakistan, the Demographic and Health Survey (DHS) of 2012-13 showed that mortality among neonates and infants was still one of highest in South Asia. Especially in the Balochistan region, where the neonatal mortality was 55 deaths per 1,000 live births and under-five mortality was 111 deaths per 1,000 live births3.\n\nMédecins Sans Frontières works in cooperation with the Ministry of Health (MoH) in two hospitals in the Balochistan province of Pakistan4. Balochistan is an unstable and vulnerable province due to historical disputes between different (ethnic) groups (Figure 1)5. One of the hospitals is located in Chaman, in the north of Balochistan near the Afghan border. The Chaman project offers services to the residents, to Afghan refugees and also to Afghans crossing the border in search of medical services. The second hospital is located in the southern region of Balochistan in Dera Murad Jamali (DMJ). This hospital offers services to the residents of Nasirabad and Jafarabad districts.\n\nAuthor: MSF UK Data Source: Natural Earth; MSF\n\nIn recent years, MSF has observed high in-hospital mortality among neonates and infants in both Chaman and DMJ hospitals. Since 2010, the recorded monthly neonatal mortality (neonatal deaths amongst all neonatal exits from the neonatal department) has exceeded 25% in both hospitals (MSF unpublished data). In order to better understand the causes for mortality and provide recommendations for clinical care, we aimed to describe the clinical characteristics of the infants under seven months admitted between 2013 and 2016 Chaman and DMJ hospitals.\n\n\nMethods\n\nThis study consisted of secondary retrospective analysis of routinely collected data in the neonatal departments of DMJ and Chaman hospitals. The clinical characteristics available for analysis were: sex, date of admission and exit (discharge or death), primary diagnosis, and outcome (i.e. death or alive). We were only able to classify patients in two age groups, under seven months and greater or equal to seven months. Thus, no specific ages were available for more refined analyses on age of infants.\n\nAll children younger than seven months old admitted to the DMJ and Chaman hospital between 2013 and 2016 were included in this study. Patients, for whom crucial data was missing such as exit date and primary diagnosis, were excluded from this study.\n\nAll data was extracted and anonymized before the analysis. We calculated an exposure time (person days) for each patient by subtracting their date of admission from the date of discharge. The primary diagnosis was categorized into sepsis, neonatal tetanus, necrotizing enterocolitis (NEC), prematurity, hypoxic-ischemic encephalopathy (HIE), maternal-fetal infection, respiratory syndromes and other. Those were chosen due to the high frequency of reported diagnosis and other contained the remaining diagnoses.\n\nThe mortality rate by month was calculated with their corresponding 95% CI and a two-months-moving-average was used to explore seasonality.\n\nWe used Poisson regression to calculate the unadjusted Rate Ratios (RRs) with their respective 95% confidence intervals (95% CI) and p-values to assess the association of each possible risk factor with the outcome of in-hospital mortality. For the primary diagnosis, we used respiratory syndromes as the reference group as it had lowest mortality rate and a sufficient number of events. A Poisson multivariable regression model was used to calculate adjusted RRs (aRR) with their corresponding 95% CI and p-values. Each patient’s exposure time was split into single day periods and this elapsed time variable was included in the Poisson models to estimate the mortality rate, rate ratios, 95% CIs and p-values for each day since admission.\n\nThe data cleaning and manipulation was done using Excel 2010, and data analysis was done using Stata IC 146. We did not merge the datasets from the two hospitals, because it would be more useful for the teams as the two hospitals contexts were different and presenting merged results may have diluted the specificities in the particular situations each team faces.\n\nEthical consideration: This was a retrospective post-hoc analysis of routinely collected clinical data; therefore, it was exempted from MSF ethical board review. The MSF-OCA medical director gave his approval for this analysis. The data in the utilized datasets did not contain individual identifiers and it was password protected and only accessible by the research team.\n\n\nResults\n\nBetween 2013 and 2016, there were 2,551 infants admitted in the Inpatient Department (IPD) of Chaman, 563 (22%) of them died (Table 1). There were more males admitted (n=1,573) than females (n=977); the male to female ratio was 1.61. The diagnosis NEC and HIE were responsible for the highest mortality rates (Table 2).\n\nNEC - necrotizing enterocolitis, HIE - hypoxic-ischemic encephalopathy\n\nIn the adjusted analysis (Table 1), the number of days since admission was strongly associated with a higher risk of mortality when compared to day eight or more days since admission. The highest risks for mortality were observed during the first five days since admission and decreased after that with each extra day of admission: at day 5 aRR=5.20 (95% CI: 3.34-8.08) and at day 8 aRR=1.28 (95% CI: 0.57-2.91). Furthermore, some primary clinical diagnoses were also associated with mortality compared to a diagnosis of respiratory syndromes, namely: NEC (aRR= 5.79; 95% CI: 3.20-10.48), prematurity (aRR=3.41; 95% CI: 2.39-4.86), HIE (aRR=3.25; 95% CI: 2.29-4.61) and suspected clinical sepsis (aRR=2.11; 95% CI: 1.50-2.98).\n\nBetween 2013 and 2015, there were 2,663 infants admitted in the IPD and 23% (n=615) died during their admission (Table 3). More males (n=1,636) were admitted than females (n=1,207); the male/female ratio was 1.59. The primary diagnosis tetanus, NEC, HIE and sepsis had the highest mortality rates (Table 4).\n\nNEC - necrotizing enterocolitis, HIE - hypoxic-ischemic encephalopathy\n\nNEC - necrotizing enterocolitis, HIE - hypoxic-ischemic encephalopathy\n\nIn the adjusted analysis (Table 3), the number of days since admission was strongly associated with death and with each extra day in the hospital this risk decreased when compared to day eight or more since admission: at day one aRR=13.38 (95% CI: 9.14-19.59) and at day eight aRR=1.72 (95% CI:0.79-3.73). In addition, patients with one of the following diagnosis had an increased risk for death versus a diagnosis of respiratory syndrome: suspected neonatal tetanus (aRR=8.50; 95% CI: 5.88-12.29), NEC (aRR=11.65; 95% CI: 4.56-29.76), HIE (aRR=4.13; 95% CI: 2.97-5.75), sepsis (aRR=3.43; 95% CI: 2.47-4.76) and prematurity (aRR=3.32; 95% CI: 2.33-4.72).\n\nWe observed an annual peak in the mortality rate between July and October in both hospitals with the mortality rate peaking in September. On average, the two-months-moving-average of the mortality rate was 40 per 1,000 person days (exposure time) for Chaman. The annual peaks in Chaman had a mortality rate that varied between 40-100 per 1,000 person days (Figure 2). For DMJ, the two-months-moving average was on average around 30 per 1000 person days. The mortality rates in the annual peaks varied between 40-70 per 1,000 person days (Figure 3). There was considerable month to month variation in mortality rates as can be observed from the 95% CIs shown in Figure 2 and Figure 3. Smoothing these crude rates using a two-month moving average still showed some evidence of an annual peaks.\n\n\nDiscussion\n\nWe found that the mortality in children under seven months age was 23% in two hospitals in Balochistan province in Pakistan between 2013 and 2016. In both hospitals, the number of days since admission was strongly associated with death: infants were more likely to die during the first 48 hours of admission, with the greatest risk at day 1 (24 to 48 hours since admission). This differs with other studies which suggest that the longer the stay in the hospital, the higher the chance to develop nosocomial infection leading to death7. Also, a longer stay in the hospital has been associated to severity of illness8.\n\nWe assume that the high mortality rate is not necessarily due to the care that MSF provides, but due to late presentation and critical state of the majority of patients in both hospitals. Anecdotally, MSF staff report that the patients are often treated with multiple antibiotics from other private health care centers. Neonatal care in private hospitals is expensive and when the financial resources of families are exhausted, parents seek help in MSF hospitals. It is known that in developing countries the private sector is preferred over public sector9, however, the provided service is debatable. We did not have any information on date of on onset of symptoms to be able to evaluate whether late presentation played a role in high mortality rates early on in their admission. Nevertheless, literature states that infant survival is lower when there is a delay in seeking health care10. Improving the health care seeking behavior will increase infant survival.\n\nThe male/female ratio in our study (1.60) was higher to the one found in the 2012-2013 DHS in Pakistan (1.04)3. This difference could be explained by son preference which has been described before in South Asia11 and/or by a reduced healthcare seeking behavior for girls by the parents in this region1,12, but we cannot be sure as this goes beyond the objective of the study.\n\nWe identified a seasonal pattern in the monthly mortality rate (per 1,000 person days) in hospitalized infants in Chaman and DMJ around September (Figure 2 and Figure 3). To our knowledge, the observed seasonal pattern has not been mentioned in the literature before. Two studies conducted in South Asia (Nepal and Bangladesh) among local populations have previously mentioned a seasonal pattern in mortality, but they found different seasonality patterns. In Nepal, the neonatal mortality rate was the highest between April and October, but the highest peak was observed in August (13). In Bangladesh, the peak of neonatal deaths was observed in November and for 1–4 year olds in the hot-wet season (July-September)13,14. However, the seasonality in mortality could be explained by seasonality of diseases. A study in Northern Pakistan showed that the highest prevalence of malaria parasites was found in infants after monsoon season (September-November)15. This similar peak (September-November) was also observed for dengue cases among admitted patients in the districts Shangla and Buner in Northern Pakistan16. In our IPD data there were no dengue diagnosis registered and the number of malaria diagnosis was very low (total 2013-2016: two in Chaman and 203 in DMJ), and did not show a seasonal pattern with increase in numbers in September.\n\nThe main limitation of this study was that it was a retrospective chart review, so there was no control in the use of data collection tools and on disease coding. We could also not explore the impact of infant age on mortality during this study. Despite the limitations, our study had the great strength of the large amount of observations around inpatient pediatric patients in this part of Pakistan, therefore the data included and the findings are highly relevant.\n\n\nConclusion\n\nThe first two days of admission are critical for infant survival in the MSF hospitals in Balochistan, an underserved area of Pakistan in terms of health care. We found an annual seasonal pattern in mortality rate by week and a male/female ratio in that was higher than the known male/female ratio of Pakistan. Further investigations are needed to establish i) if the cause of this male/female ratio differences is gender differences in access to care or an actual difference in burden of disease by gender and ii) the reasons behind these annual peaks in mortality rate by week. We recommend targeting efforts on increasing quality of care during the first days of admission and to allocate resources accordingly, and also taking into account the seasonal pattern.\n\n\nEthical considerations\n\nThis was a posteriori analysis of routinely collected clinical data; therefore, it was exempted from ethical board review. The MSF-OCA medical director gave his approval for this analysis. The data in the utilized datasets did not contain individual identifiers and it was password protected and only accessible by the research team.\n\n\nData availability\n\nThe nature of MSF operations and target populations are such that data collected often involves highly Sensitive Data. Recipients, who wish to access any MSF Datasets that include Personal Data and/or Human Samples, must secure ethical clearance from competent ethical authorities and of MSF ERB. If a reader wants to access the data he/she can find more information in the MSF Data Sharing Policy (http://hdl.handle.net/10144/306501).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to acknowledge the MSF and MoH staff in the DMJ and Chaman hospitals, they work hard every day to improve the health of these populations, without their effort this study would not have been possible. We would also like to thank the local populations for welcoming the MSF and MoH staff.\n\n\nReferences\n\nLawn JE, Kerber K, Enweronu-Laryea C, et al.: 3.6 million neonatal deaths--what is progressing and what is not? Semin Perinatol. 2010; 34(6): 371–86. PubMed Abstract \| Publisher Full Text\n\nUnited Nations: The Millennium Development Goals Report. United Nations. 2015: 72. Reference Source\n\nNIPS: Pakistan Demographic and Health Survey 2012-13. 2013; 392. Reference Source\n\nDoctors without Borders, the Médecins Sans Frontières: International Activity Report 2015. 2016; 100. Reference Source\n\nKupecz M: PAKISTAN’S BALOCH INSURGENCY: History, Conflict Drivers, and Regional Implications. Int Aff Rev. 2012. Reference Source\n\nStataCorp: Stata Statistical Software: Release 14. 2015. 2015. Publisher Full Text\n\nSingh-Naz N, Sprague BM, Patel KM, et al.: Risk factors for nosocomial infection in critically ill children: A prospective cohort study. Crit Care Med. 1996; 24(5): 875–8. PubMed Abstract \| Publisher Full Text\n\nFiser DH, Tilford JM, Roberson PK: Relationship of illness severity and length of stay to functional outcomes in the pediatric intensive care unit: a multi-institutional study. Crit Care Med. 2000; 28(4): 1173–79. PubMed Abstract \| Publisher Full Text\n\nShaikh BT, Hatcher J: Health seeking behaviour and health service utilization in Pakistan: Challenging the policy makers. J Public Health (Oxf). 2005; 27(1): 49–54. PubMed Abstract \| Publisher Full Text\n\nLassi ZS, Middleton PF, Bhutta ZA, et al.: Strategies for improving health care seeking for maternal and newborn illnesses in low- and middle-income countries: A systematic review and meta-analysis. Glob Health Action. 2016; 9: 31408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta MD, Zhenghua J, Bohua L, et al.: Why is Son preference so persistent in East and South Asia? a cross-country study of China, India and the Republic of Korea. J Dev Stud. 2003; 40(2): 153–187. Publisher Full Text\n\nJehan I, Harris H, Salat S, et al.: Neonatal mortality, risk factors and causes: A prospective population-based cohort study in urban Pakistan. Bull World Health Organ. 2009; 87(2): 130–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes MM, Katz J, Mullany LC, et al.: Seasonality of birth outcomes in rural Sarlahi District, Nepal: a population-based prospective cohort. BMC Pregnancy Childbirth. 2014; 14: 310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker S, Weng S: Seasonal patterns of deaths in Matlab, Bangladesh. Int J Epidemiol. 1998; 27(5): 814–23. PubMed Abstract \| Publisher Full Text\n\nZeb J, Sayed Khan M, Ullah H, et al.: Epidemiology of Plasmodium Species and Prevalence of Malaria on the Basis of Age, Sex, Area, Seasonality and Clinical Manifestation in the Population of District Lower Dir, Khyber Pakhtunkhwa, Pakistan. World J Zool. 2015; 10(2): 147–152. Reference Source\n\nKhan J, Munir W, Khan B, et al.: Dengue outbreak 2013: Clinical profile of patients presenting at DHQ Burner and THQ Shangla, Khyber Pakhtunkhwa, Pakistan. Immun Dis. 2015; 3: a11. Reference Source" }	[ { "id": "50282", "date": "08 Jul 2019", "name": "Babar Tasneem Shaikh", "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 is an interesting approach to study such data in retrospective fashion. Findings on most common newborn or infants’ illnesses and their outcomes (discharge or mortality) in Pakistan are well known and covered under various national surveys also. PDHS and MICS are two such examples besides various individual and organizational work published in recent years.\nThe paper under consideration has used PDHS 2012-13 as a baseline and has attempted to make a case for two sites in Balochistan. The main thrust is that province under-performs vis-à-vis national statistics. This is not a new revelation and is a well-known and documented fact for many decades. With new PDHS 2017-18 released, this paper becomes redundant. Secondly, the causes of hospital admission or diagnoses found in infants are also chronologically known. Only thing which could have made this paper more interesting is adding the work of MSF which is being done at these two sites to address the issue of neonatal morbidity and mortality and the details of interventions which are being introduced at hospital or community level and then results shown with a significant difference made.\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": "4753", "date": "12 Feb 2020", "name": "Babette van Deursen", "role": "Author Response", "response": "Dear Mr Shaikh, Thank you for your revision of our article and your very useful comments. We believe that they have helped us to improve our paper. To start with your comment on the PDHS 2017-2018, we would like to clarify that we were not aware of the new version when we submitted this manuscript. When we were drafting the manuscript last year, it was not published yet. So, your suggestion is highly pertinent and helpful. We think it is indeed better to use the newest results instead of the PDHS of 2013. We have amended accordingly. However, we still think our manuscript is not redundant since the PDHS does not include data on seasonality patterns in mortality, nor on time to death from admission. So our manuscript provides extra information on these two topics. Regarding your point about describing the activities of MSF, we agree that it would be very interesting to elaborate more on this. Unfortunately it would be very difficult now for us to get these details as this was not included in our original request to the MSF research committee and to the team on the ground. We believe that your comments and suggestions have helped us to improve the context information and the discussion of our results. We very much appreciate your time and comments. Many thanks. Yours sincerely,Babette van DeursenAdjustments: Introduction: We can use the new data from PDHS 2017-2018 instead of the old PDHS 2013 on the mortality numbers. Discussion: New ratio male/female ratio for under-five 1.02 (PDHS 2017-2018). Small difference between female/male treatment seeking behavior, especially for fever. However, these small differences do not fully explain our found difference in health seeking behavior between boys and girls." } ] }, { "id": "51351", "date": "26 Jul 2019", "name": "Eva Leidman", "expertise": [ "Reviewer Expertise Epidemiology with focus on humanitarian emergencies." ], "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 use routine data from two MSF hospitals in Pakistan to explore factors associated with in-hospital mortality among infants and neonates. As such the manuscript presents interesting analysis of an important research question. The authors nicely characterize the benefits and limitations of the retrospective data (e.g., lack of age data) used in the presented manuscript.\nThe manuscript would benefit from further details pertaining to the statistical analysis, as well as justification of the model selection. Additional details should include analysis performed to assure the assumptions of the Poisson model hold (e.g., measure of dispersion), and clarity on how the offset was modeled.\nAdditionally, as the key finding of the manuscript relates to differences in mortality associated with days since admission, (descriptive) analysis comparing characteristics of children with exits in the first 48 hours to the reference would be strengthen the manuscript, and help explore implications of the regression findings. Finally, global tests for month of admission (seasonality) appear only significant for Chaman (not DMJ). Discussing this distinction would also strengthen the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "51353", "date": "06 Sep 2019", "name": "Sharif Ismail", "expertise": [ "Reviewer Expertise Health systems and policy 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\nThank you for the opportunity to review this paper, which presents data from two MSF hospitals in Pakistan to explore clinical factors associated with mortality among children admitted to these facilities. The analysis of seasonality in particular is an interesting and novel feature of this analysis although it is notable that differences between the two sites in this regard are not commented on in the manuscript.\nThere are some important ways in which the work could have been strengthened. For example:\nThe choice of a 7-month age cut off in defining the patient group is unusual as it corresponds neither to traditional definitions of the neonatal nor infant periods. It would have been helpful to know exactly why it would possible only according to the 7-month cut off and to have read a clearer explanation of the implications of this limitation for the analysis - and more on data limitations overall would have been useful.\n\nTables 2 and 4 aggregate diagnostic categories differently - why is this? Were no cases reported against the other categories? Separating out categories in the same way would have aided qualitative comparisons between the two sites.\n\nThe authors state that a pooled analysis across the two facilities was not practically useful to the teams. This is fine - but it would then have been helpful to include more data (if available) to test to account for observed differences - especially in mortality within the first 48 hours of admission. Were any triage assessment measures on admission available to include?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-954
https://f1000research.com/articles/8-908/v1	20 Jun 19	{ "type": "Software Tool Article", "title": "Visualization of drug target interactions in the contexts of pathways and networks with ReactomeFIViz", "authors": [ "Aurora S. Blucher", "Shannon K. McWeeney", "Lincoln Stein", "Guanming Wu", "Aurora S. Blucher", "Shannon K. McWeeney", "Lincoln Stein" ], "abstract": "The precision medicine paradigm is centered on therapies targeted to particular molecular entities that will elicit an anticipated and controlled therapeutic response. However, genetic alterations in the drug targets themselves or in genes whose products interact with the targets can affect how well a drug actually works for an individual patient. To better understand the effects of targeted therapies in patients, we need software tools capable of simultaneously visualizing patient-specific variations and drug targets in their biological context. This context can be provided using pathways, which are process-oriented representations of biological reactions, or biological networks, which represent pathway-spanning interactions among genes, proteins, and other biological entities. To address this need, we have recently enhanced the Reactome Cytoscape app, ReactomeFIViz, to assist researchers in visualizing and modeling drug and target interactions. ReactomeFIViz integrates drug-target interaction information with high quality manually curated pathways and a genome-wide human functional interaction network. Both the pathways and the functional interaction network are provided by Reactome, the most comprehensive open source biological pathway knowledgebase. We describe several examples demonstrating the application of these new features to the visualization of drugs in the contexts of pathways and networks. Complementing previous features in ReactomeFIViz, these new features enable researchers to ask focused questions about targeted therapies, such as drug sensitivity for patients with different mutation profiles, using a pathway or network perspective.", "keywords": [ "targeted therapy", "drug interaction visualization", "Reactome", "biological pathway", "functional interaction network", "Boolean network", "constrained fuzzy logic modeling", "systems pharmacology" ], "content": "Introduction\n\nThe overarching aim of precision medicine is to provide patients with the best choice of therapy based on their individual genetic and/or epigenetic alterations. For cancer and other complex diseases, a key strategy often used is to harness these alterations to stratify patients into subgroups to receive the most appropriate targeted therapy. However, we currently face many challenges such as identifying rigorous biomarkers to guide treatment decisions, rationally designing combination therapies for improved efficacy, reducing therapeutic toxicity and side effects, and managing both intrinsic and adaptive drug resistance to therapy1–3.\n\nPathway and network analysis approaches to understanding the interactions between genetic variation and therapeutic response offer researchers the unique ability to understand these interactions in their rich biological context. While pathways are groups of biological entities that are connected together to carry out specific functions or biological processes, networks of entities are usually constructed in a systems-manner, covering many pathways at the same time. In cancer, shared oncogenic pathways in patients may provide an opportunity to stratify and identify therapeutic options. For instance, targeting kinases in the MAPK and PI3K/AKT signaling pathways has been very successful for cancer treatment due to the high prevalence of patient mutations in these pathways3,4. Note that patients may share the same pathway-level dysregulation that is acquired through alterations in different genes in the pathway or in a set of genes that span multiple pathways. While the first scenario is well-suited to pathway analysis, the second would benefit from network methods that encompass across multiple pathways. Both scenarios provide opportunities for biomarker discovery to inform patient stratification, target prioritization, or treatment assignment by applying pathway and network approaches5.\n\nPathway- and network-based approaches have also been successfully used in other aspects of targeted therapy. For example, they have been used successfully in prediction of drug side effects and in explaining critical toxicity issues in failed drugs6,7. They have also been used to unravel the drug resistance mechanisms. Two notable mechanisms of drug resistance in tumors include gatekeeper mutations in direct drug targets (such as those often found in oncogenic kinases) and mutations in non-drug targets that enable bypass resistance pathways used by tumor cells in response to drug-induced inhibition of pathways8. In the second case, mutations in proteins other than the direct target of a drug can confer resistance by activating downstream and/or parallel pathways9.\n\nIn each of these applications, it is imperative to be able to visualize the intended effect of drugs on pathways through its designated targets. Currently available resources that offer such visualization include ConsensusPathDB10 and PharmGKB11. ConsensusPathDB aggregates functional interactions including protein interactions, gene-gene interactions, metabolic network interactions, and drug-target interactions from public resources10. With respect to drug-target interactions, ConsensusPathDB allows for selection of interactions for visualization but does not provide functionality for overlaying drugs to a pathway of interest. PharmGKB provides a curated drug-centric view of pathways that aggregates information for a particular drug’s targeted pathways, according to the disease for which the drug is designed11. However, these resources do not provide a means of selecting and filtering drug-target interaction evidence and do not overlay drug-target information to all pathways.\n\nIn addition to visualizing both primary and secondary or “off” targets of drugs in the contexts of pathways and networks, we also need to be able to investigate the effect of perturbation either via one drug or a combination of drugs. For this purpose, we need to perform pathway mathematical modeling. In recent decades, several pathway modeling approaches have been developed, including models leveraging ordinary differential equations (ODEs)12, Petri-Nets13, flux-based analysis (FBA)14, probabilistic graphical models (PGMs)15, and Boolean Networks16,17. ODE modeling is the most sophisticated because it is able to generate both quantitative and dynamic behaviors of model entities. However, lack of initial concentrations and key parameters often limits the application of this approach to small pathways and networks. While Petri-Net and FBA approaches have been used in metabolic network modeling, the sensitivity of these methods to the underlying network structures makes it difficult to apply them to signal transduction pathways because of the incompleteness in many signaling networks due to current limited knowledge. PGMs have been used to integrate multiple 'omics data types together for pathway impact analysis15. However, the need to learn model parameters makes this approach difficult, if not impossible, in many applications due to small sample sizes and/or incomplete network structure. Furthermore, PGMs cannot handle dynamic modeling well since inference results from these models reflect \"beliefs\" for states of model variables and do not involve kinetic behavior as produced by dynamic modeling.\n\nBoolean network modeling is one of most widely used modeling approaches and has been applied to several large-scale network modeling efforts due to its simplicity and high efficiency. Originally introduced by Kauffman18, Boolean networks provide a method of modeling biological pathways and networks using Boolean variables. Each biological entity in a Boolean network can be in either the active (1) or inactive (0) state, which is determined by logic-based rules involving the network’s functional interactions. A two-state-based Boolean network can be extended to a fuzzy logic model through introduction of continuous variables with values between 0 and 1, inclusively, and applied to biological use cases that involve continuous variables19.\n\nReactomeFIViz20, a Cytoscape21 app, was developed to perform pathway- and network-based data analysis and visualization for cancer and other complex disease data using Reactome pathways22 and its complementary resource, the Reactome Functional Interaction (FI) network23. Recent enhancements to ReactomeFIViz provide features for visualization of drug-target interactions for approved clinical drugs in the contexts of pathways and networks and modeling the effect of drug action on the pathway activities (Figure 1). These features offer a versatile and integrative platform to interrogate the full range of drug-target-pathway-network interactions. In this report, we describe these new features and show their utility from different perspectives using a series of practical examples.\n\nUsing ReactomeFIViz, researchers can visualize drug-target interactions according to strength of supporting binding assay evidence, visualize drug-target interactions in the contexts of pathways and functional interaction networks, and perform Boolean Network modeling to investigate the impact of drugs on pathways.\n\n\nFeatures with Use Cases\n\nReactomeFIViz provides features for visualization of drug-target interaction information for 171 FDA-approved cancer drugs from the Cancer Targetome24 and for 2,102 world-wide approved drugs from DrugCentral25,26, facilitating researchers in investigating supporting evidence for interactions between a drug and all its targets. These features address a major bottleneck in precision therapy research - the lack of rigorous and transparent supporting information on drugs and their targets. While it is well-established that many approved drugs are promiscuous and interact with many targets, it is more difficult to obtain and visualize reliable information about such additional targets27,28. Resources such as the Cancer Targetome and DrugCentral provide thorough drug-target information, but do not provide visualizations for these drug-target interactions. By adding functionality for researchers to visualize drug-target interactions, ReactomeFIViz empowers investigation of drug-targets in a supporting evidence-based manner.\n\nAll drugs collected in the Cancer Targetome and DrugCentral can be accessed within ReactomeFIViz. For each drug, target interaction evidence can be filtered according to strength of the supporting assay values and displayed as either a table or as a histogram (Figure 2). This allows the user to assess drug-target relationships on the basis of supporting evidence. For example, the histogram in Figure 2 for target assay values for the drug sorafenib shows that there are many potential targets with assay values under 100 nM. Targets that have multiple assay values under 100 nM include FLT3, RET, KIT, RAF1, and BRAF. Additional targets with very low assay values include DDR1, DDR2, FLT4, and KDR, among others. Overall there is evidence across multiple assay types to support sorafenib interactions with a variety of targets. Sorafenib is often referred to as a “multi-kinase” inhibitor, and promiscuity (interactions with many targets) has been well noted in the literature29,30. The ability to display and threshold the full range of supporting evidence is a key requirement for researchers engaged in drug discovery efforts. Some applications, such as the nomination of compounds for drug screens, will require very strong binding assay evidence and would benefit from being able to compare results across multiple assay types. Other applications, especially those that perform in silico modeling, may have less stringent requirements for binding assay values. For instance, researchers interested in exploring compounds for drug repurposing may want to look at more weakly binding compounds as a starting place before further optimization.\n\nSorafenib interacts with many targets, even when restricting to target interactions supported by binding assay evidence < 100 nM.\n\nDepending on the application, different perspectives on drug-target-pathway interaction data are necessary. If we are focused on investigating a particular drug or a small number of related drugs, we would want to explore targets and pathways. For instance, if we want to investigate off-target or toxic effects of a certain drug, we may want to consider all possible targets and pathways with which the drug interacts. In such a scenario, we can look up all the target interactions for a particular drug and map them to pathways. Furthermore, performing enrichment analysis identifies pathways with a significant number of targeted entities, suggesting pathways most perturbed by the drug. The top enriched pathways for sorafenib targets with supporting assay values <= 100 nM are shown in Table 1. Sorafenib is a receptor tyrosine kinase inhibitor, which is known experimentally to interact with a variety of targets. These targets are involved in several signaling pathways. For instance, we can see (Table 1) that many of sorafenib’s targets are involved in the RAF/MAP kinase cascade as well as other pathways involving VEGF signaling and PIP3/AKT signaling. Examining the full range of pathways targeted by a drug allows us to better understand the drug’s mechanism of action for both efficacy and side effects.\n\nTargets for the drug sorafenib with supporting assay values < 100 nM were retrieved from the Cancer Targetome and then mapped to pathways. Pathway enrichment analysis was performed using a binomial test and p-values were FDR-corrected for multiple testing. The table was generated by ReactomeFIViz. Only pathways having FDR <= 0.01 are listed here.\n\nAlternatively, we may start with a pathway of interest and investigate all drugs that target components of the pathway within a selected affinity range. This perspective allows a broad view for assessing how confident we may be able to find a drug targeted to a particular pathway, as many pathways have only weakly or no interacting drugs24. Figure 3 highlights drugs targeting KIT in the pathway “SCF-KIT signaling” (https://reactome.org/content/detail/R-HSA-1433557), which are retrieved by using the “Fetch Cancer Drugs” feature in ReactomeFIViz to visualize drug-target interactions collected in the Cancer Targetome. As displayed in Figure 3, KIT is targeted by ten different drugs, which are all kinase inhibitors: erlotinib, imatinib, bosutinib, sunitinib, pazopanib, dasatinib, sorafenib, axitinib, vandetanib, and nilotinib24,29, and supported by assay values less than 100 nM. This view suggests a list of potential drug candidates for researchers to perturb the activity of the SCF-KIT signaling pathway.\n\nDrug-target interactions were fetched from the Cancer Targetome and supported with multiple assay types having values <= 100 nM.\n\nOverlaying drug-target interactions onto a pathway reveals potential drug action targets in the pathway. However, the topological structure of a pathway may be complicated and contain one or more feedback loops. This makes it difficult to predict the dynamic effect of a drug on pathway activity by inspection only. To assist researchers in performing computational modeling of drug-induced pathway perturbation, we developed an automated approach to convert Reactome pathways into Boolean networks (BNs). These BNs were then subject to constrained fuzzy logic simulation31,32 to model a drug’s perturbation on the activity of a particular pathway. We conducted two constrained fuzzy logic simulations: a reference simulation without considering drug action, and a perturbation simulation considering drug action. To model drug action within the simulation, we mapped drug-target interaction affinity values as “activation” or “inhibition” strength according to the drug’s action mechanism and its assay values collected in the drug data sources. We then used this information to modify the transfer functions related to the drug's target(s) in the fuzzy logic model. Finally, we calculated the relative impact scores for individual entities in the pathway by comparing the model that includes drug effects to the one that does not. For details, see Methods.\n\nTo illustrate this capability, we take the example of modeling the perturbation induced by the kinase inhibitor sorafenib on the “Signaling by SCF-KIT” pathway. Stem cell factor (SCF), together with its receptor, c-KIT, a tyrosine kinase receptor, regulates pathways related to proliferation, migration, survival, and differentiation of multiple cell types33. These regulated pathways include RAF/MAP kinase signaling, PIP3-activated AKT signaling, and the JAK/STAT signaling pathway. The Reactome-annotated pathway “Signaling by SCF-KIT” includes a set of linked reactions that produce entities activating these regulated pathways.\n\nDrugs targeting proteins involved in SCF-KIT signaling have been under active study for many years34. Evidence shows that sorafenib binds strongly to c-KIT (Figure 2 and Figure 3) with a minimum binding constant of 16 nM (KD) according to the Cancer Targetome. We performed four constrained fuzzy logic simulations to study the perturbation of sorafenib on the activity of a complex called “p-STAT dimers”, which is one of the major outputs of the SCF-KIT pathway. “p-STAT dimers” activates expression of genes regulated by STAT proteins and is produced by the reaction “Disassociation and translocation of STATs to the nucleus”. This reaction, together with three other reactions, “Recruitment of STATs”, “Phosphorylation of STATs”, and “Dimerization of STATs”, forms a loop, providing a way to produce activated STAT dimer by recycling the “p-JAK2:SFKs:p-KIT\" complex (Figure 4). The four simulations were:\n\nThe entry point of the loop and “p-STAT dimers”, together with four reactions forming the loop, are highlighted in light blue. The reactions are labeled with names in the light-grey boxes. The target of drug sorafenib, KIT protein, is in the upstream of this loop and not shown here.\n\n1. Default setup provided by ReactomeFIViz without sorafenib (reference_1 as unperturbed state);\n\n2. Default setup with sorafenib (sorafenib_1 as drug-perturbed state);\n\n3. Default setup with a reduced initial value of PRKCA [cytosol] (https://reactome.org/PathwayBrowser/#/R-HSA-1433557&SEL=R-HSA-58196&PATH=R-HSA-162582,R-HSA-9006934) from 1.0 to 0.5. No sorafenib was applied (reference_2 as PRKCA-perturbed state). PRKCA is protein kinase C alpha, which phosphorylates KIT and therefore inhibits KIT’s kinase activity (https://reactome.org/PathwayBrowser/#/R-HSA-1433557);\n\n4. Same as in 3) except that sorafenib was applied (sorafenib_2 as drug/PRKCA-perturbed state).\n\nIn Figure 5, we show the activity (labeled as Logic Fuzzy Value in the y-axis) of “p-STAT dimers” over time steps in the simulation. It is well known that pathways having feedback loops usually show periodic cycle patterns or attractors for activities of entities involved in the loops35. As expected, we saw these attractors in all four simulations. In the first drug perturbation modeling (reference_1 and sorafenib_1, Figure 5A) with the initial value of PRKCA equal to 1.0 as fully activated, the application of sorafenib did not notably perturb the activity of “p-STAT dimers” (relative impact score = 8.63E-4), most likely because the loop used to produce “p-STAT dimers” was not affected by sorafenib under this simulation’s condition. However, by reducing the initial value of PRKCA from 1.0 to 0.5, the cycle attractor of “p-STAT dimers” changed from (0, 1, 0, 0, 1, 1) to (0.5, 1, 0.5, 0.5, 1, 1) in the absence of sorafenib (simulation reference_2, Figure 5B), increasing the lower activity in the attractor. Reduction of the initial value of PRKCA brought down the activity of “p-KIT (S741,746):PKC alpha”, the inhibitor of the reaction, “Autophosphorylation of KIT”, resulting in an increase of the activity of “p-JAK2:SFKs:p-KIT complex” via several intermediate reactions. “p-JAK2:SFKs:p-KIT complex” is the entry point of the loop that produces “p-STAT dimers” (Figure 4). Increasing the activity level of this complex levels up the p-STAT dimers, thereby increasing the lower value of its cycle attractor. Intriguingly, the application of sorafenib abolished the effect of the reduction of PRKCA’s initial value, bringing down the lower value of the cycle attractor from 0.5 to 0.0024, the same value when PRKCA’s initial value was set at 1.0. Hence, sorafenib had a much stronger perturbation when PRKCA’s initial activity was lower (relative impact score = -0.17). For the detailed pathway annotation, see Figure 6 and also refer to the Reactome pathway diagram: https://reactome.org/PathwayBrowser/#/R-HSA-1433557.\n\nPanel A: Simulations conducted with the default setup provided by ReactomeFIVIz. Logic fuzzy values for two simulations, reference_1 and sorafenib_1, are almost the same except bottom values starting from step 18: 0.0 for reference_1 and 0.0024 for sorafenib_1 (see the insert for an example). Panel B: Same as in Panel A except the initial value of PRKCA was reduced from 1.0 to 0.5. Logic fuzzy values prior to time step 18 are the same for both reference and sorafenib simulations. The results were exported from ReactomeFIViz and plotted in Microsoft Excel.\n\nThe above comparison analysis of the simulation results demonstrates the importance of mathematical modeling, even without experimentally measured parameters, by predicting that alterations in the PRKCA gene may impact the effects of the drug sorafenib on SCF-KIT signaling. For instance, a loss-of-function mutation in the PRKCA gene could influence how sorafenib affects the SCF-KIT pathway. Alternatively, another drug that inhibits PRKCA could be used in combination with sorafenib to potentially impact the pathway in a different manner than with sorafenib alone. This level of mechanistic and dynamic investigation is not possible by merely overlaying drug-target interactions onto the pathway.\n\nWe also performed analysis for another output of this pathway, “PI(3,4,5)P3” (https://reactome.org/PathwayBrowser/#/R-HSA-1433557&SEL=R-ALL-179838) (Extended data: Figure S1), which was annotated as an activator for pathway “PIP3 activates AKT signaling”. We observed similar behavior as in the case of “p-STAT dimers”: reducing the initial value of PRKCA increased the activity of “PI(3,4,5)P3”. The application of sorafenib modestly increased PI(3,4,5)P3's activity using default PKCA activity level (1.0), but reduced its activity when PRKCA’s activity was reduced. However, the relative impact scores for PI(3,4,5)P3 are much larger compared to the case of the p-STAT dimers described above, most likely because this entity has a converged single stable activity, in contrast to p-STAT dimer’s cycle attractor.\n\nWe’d like to emphasize that the above simulations were performed using a set of initial values and transfer function parameters that we developed to ensure simulations with converged solutions. In reality, the actual tumor cell conditions will likely differ from the parameters we used. ReactomeFIViz provides a set of intuitive user interfaces for users to try different parameters in simulation. Future work for this modeling framework will include approaches for estimating and learning these parameters based on large scale omics datasets. We hope to address this daunting issue soon.\n\nTo assist researchers in investigating drug impact on pathway activities in a systematic way, ReactomeFIViz provides a feature to highlight entities based on relative impact scores. Figure 6 shows SCF-KIT Signaling pathway highlighted according to the relative impact scores with PRKCA’s initial value equal to 1.0 (Figure 6A) and 0.5 (Figure 6B) for sorafenib.\n\nA: Relative impact scores for sorafenib with PRKCA’s initial value equal to 1.0 as fully activated. B: Same as A except that the initial value of PRKCA was reduced from 1.0 to 0.5.\n\nA network perspective unites multiple pathways together, allowing a bird’s eye view of drug-target relationships. This perspective is especially important when considering drugs with targets that span multiple pathways or considering potential crosstalk between pathways (e.g. Table 1). Projecting drug-target interactions onto a network helps prioritization of targets, targeted pathways, and targeted therapies. Here, we use the TCGA ovarian cancer data36 as an example to show the utility of this network perspective for prioritizing targeted therapies for ovarian cancer.\n\nThe Reactome functional interaction (FI) network was constructed by extracting functional relationships from manually curated pathways in Reactome and several other pathway databases, and then combining them with predicted interactions based on a machine learning approach, Naïve Bayes Classifier23. Using genes mutated in TCGA ovarian cancer samples, we constructed a FI network, where each node represents a gene mutated in at least 5 patients in the TCGA cohort, and each edge represents a functional interaction between two genes.\n\nCombining the TCGA ovarian cancer FI network with the Cancer Targetome reveals that there are 18 different cancer drugs in the Cancer Targetome that potentially interact with target proteins in the ovarian cancer set, at affinity values <= 100 nM. One strategy for prioritizing drugs interacting with members of this network would be to prioritize drugs targeting proteins that are also members of multiple significantly enriched pathways for the ovarian cancer cohort. For instance, epidermal growth factor receptor (EGFR) may be prioritized as a target for ovarian cancer. It has roles in multiple pathways that are significantly enriched for mutated genes, including “L1CAM interactions”, “Focal adhesions”, “Calcium signaling pathway”, “PI3K-AKT signaling pathway”, and “HIF-1 signaling pathway” (Extended data: Table S1). EGFR is targeted by both monoclonal antibody drugs and kinase inhibitors37. 14 of the 18 drugs that interact with targets in the network bind to EGFR (Figure 7). EGFR is a member of the well-studied ERBB protein family and is a target of interest for ovarian cancer37. Many studies have shown encouraging results for the effect of EGFR and other ERBB-family inhibitors on ovarian cancer cell lines38. However, despite promising preclinical evidence, inhibitors targeting the ERBB signaling have up to this point shown little efficacy in patient clinical trials38. Current efforts in this area are focused on the use of ERBB-family inhibitors in combination with cytotoxic or targeted therapies39,40.\n\nOnly part of the FI network was shown here for drugs targeting protein products of genes in the network. Network nodes are genes mutated in at least 5 TCGA samples, where size of the node indicates the number of samples with the mutated gene. Black edges are FIs between genes, blue edges drug-target interactions with <= 100 nM supporting assay values, dashed edges predicted FIs, solid edges extracted from annotated FIs, -> activation or catalysis FIs, -\| inhibition FI, - FIs extracted from complexes or inputs of reactions. EGFR, TNIK, PAK3 and sunitinib malate are highlighted in yellow.\n\nAn alternative strategy for prioritizing drugs interacting with this FI network would be to select drugs interacting with targets in multiple, disjoint pathways to potentially achieve a greater coverage of key genes in the network. For instance, the drug sunitinib malate targets both “TRAF2 And NCK Interacting Kinase” (TNIK) and “P21 (RAC1) Activated Kinase 3” (PAK3) (Figure 7), which are individually involved in different pathways enriched by mutated genes (Extended data: Table S1).\n\n\nDiscussion\n\nPathway and network-based approaches for precision medicine offer a holistic view of drug action, providing biological contextualization for drug-target interactions. We have developed a user-friendly, integrative software platform by enhancing a popular Cytoscape app, ReactomeFIViz, for the community to perform pathway and network-based drug-target interaction visualization, modeling, and analysis.\n\nReactome22 is one of the most popular biological pathway databases, providing high quality manually curated biological pathways that cover over 50% of known human genes. Pathways in Reactome are annotated according to biochemical reactions, which are connected together to form networks. We developed a scheme to automatically convert reaction-based pathways in Reactome into Boolean networks and then perform constrained fuzzy logic based simulation31,32. Our approach is generic and can be adopted for other reaction-based pathway databases (e.g. Panther Pathways41) and pathways provided in the BioPAX format42, the community standard for pathway data exchange.\n\nFuture improvements to our Boolean network-based fuzzy logic modeling approach will find a better measure to quantify the impact of drug perturbation on entity activities. The current relative impact score may be too sensitive when the area under curve of the fuzzy logic values vs time steps generated from the reference simulation is very close to 0, as shown for the PIP3 example (Extended data: Figure S1). Future improvements will also include expansions for learning and/or optimizing parameters based on omics data and improving handling of entity set members for tissue-specific simulation by annotating tissue-specific information. Furthermore, collaboration with bench scientists will help to validate and refine the computational models.\n\nAn unmet need in understanding drug resistance is the need for additional mechanistic information on how mutations in drug targets may affect drug-target binding, one of the major causes of resistance8. The major drug-target interaction data resources, including Cancer Targetome and DrugCentral, have not collected this type of information yet, and ReactomeFIViz cannot perform pathway modeling for this type of ‘on-target’ resistance8. We are working on introducing features to ReactomeFIViz to help users visualize protein mutant locations in protein-protein interaction 3D structures by utilizing the structural data generated by Mechismo, a software tool used to infer 3D structural information and functional impact of structural variants on protein-protein and protein-chemical interactions43,44. We plan to integrate these features with drug-target interactions once this type of data is available in the future.\n\nA second challenge in understanding drug resistance is pathway-level mechanisms of resistance. Bypassing drug-targeted pathways is another common mechanism for cancer drug resistance8,9. An improved understanding of pathway redundancy and crosstalk inside cells will help us to uncover the mechanisms driving this type of drug resistance. However, the majority of pathway databases, including Reactome, do not yet provide a systematic pathway and network map for visualization and modeling of pathway crosstalk. We envision an integrative systems pathway map in the future, including signaling transduction, gene regulation, and metabolism, that will aid in this type of analysis.\n\nOverall, visualization of drug-target interactions in the contexts of pathways and networks and in silico modeling of drug perturbation allow for prioritization of drugs with greater promise for effective pathway targeting in cancer cells. This is particularly valuable for drug combination discovery, as the number of possible drug combinations for even a modestly sized set of monotherapies can be intractable to test experimentally. Promising leads from perturbation analysis can be prioritized for bench testing. Furthermore, the examples discussed here are applicable for drugs outside the cancer domain. We foresee a routine adoption and use of pathway and network-based prioritization methods for repurposing drugs from one therapeutic domain into other domains45.\n\n\nMethods\n\nThe Cancer Targetome24 aggregates drug-target interactions from four public databases: DrugBank46, Therapeutic Targets Database47, the IUPHAR/BPS Guide to Pharmacology48, and BindingDB49. This resource provides interaction information across 171 FDA-approved cancer drugs, 880 protein targets, and over 6800 interactions between these drugs and targets. Each drug interaction is accompanied by supporting evidence across three tiers24: parent database, PubMed IDs for literature references, and experimental assay values. Experimental assay values include IC50, EC50, KD, and Ki values. Each drug-target interaction may be supported by multiple types of assay values. The companion supporting evidence allows for assessment of putative drug-target interactions in an empirical data-driven manner, in particular, for consideration and inclusion of secondary or “off” targets of drugs that may play an important role in a drug’s effects on a particular pathway. For more information, see 24. The version currently supported in ReactomeFIViz was developed in 2016.\n\nThe DrugCentral25 database collects extensive drug information, including but not limited to compound structure, active ingredients, and mechanism of action for approved drugs from the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and the Japan Pharmaceuticals and Medical Devices Agency (PMDA). Target information in DrugCentral is mined from bioactivity resources such as ChEMBL and IUPHAR, and may also be supplemented with manually curated target information from drug approval labels. Overall, DrugCentral (version released in August, 2017) includes information for 2102 compounds, 1550 targets, and 13,407 drug-target interactions. The drug-target interaction file supported by ReactomeFIViz, drug.target.interaction.08292017.tsv, was downloaded from the DrugCentral web site (http://drugcentral.org) in 2017.\n\nFor the functional interaction network in Figure 7, we used the Gene/Mutation Set Analysis feature in ReactomeFIViz to create a network with genes mutated in at least 5 samples in the TCGA Ovarian Cancer dataset36. Pathway enrichment p-values in ReactomeFIViz were calculated using the binomial test and were false discovery rate (FDR)-corrected based on the Benjamini-Hochberg approach50.\n\nIn Reactome, the biochemical reaction is the basic unit for pathway annotation. Pathways are annotated as a set of biochemical reactions that are linked together. We developed a scheme to automatically convert Reactome pathways into Boolean networks (Figure 8) by adopting our previous approach used to convert Reactome pathways into probabilistic graphical models20 based on the PARADIGM approach15. A typical reaction in Reactome has one or more inputs, one catalyst, one or more activators, one or more inhibitors, and one or more outputs. A logical “AND” relationship was created among inputs, the catalyst, and activators. Inhibitors were negated and then added into this “AND” relationship. To make the relationship simple, if multiple outputs, activators, or inhibitors are annotated for a reaction, we introduced accessory nodes for them. Therefore, a typical reaction in Figure 8A was converted into a set of Boolean relationships (· for AND, + for OR, ! for NOT):\n\nA: Convert a typical Reactome reaction into a set of Boolean relationships. B: Convert a complex into an AND relationship between complex subunits and the complex. C: Convert an EntitySet into an OR relationship between set members and the set.\n\ninput1 · input2 · catalyst · activator_acc · !inhibitor_acc = output_acc\n\nactivator1 + activator2 = activator_acc\n\ninhibitor1 + inhibitor2 = inhibitor_acc\n\noutput_acc = output1\n\noutput_acc = output2\n\nReactome has annotated many complex assembly reactions. However, some complexes are annotated directly for reactions without explicit description of how subunits are assembled together. We expanded a complex like this into an “AND” relationship among its subunits and generated the following Boolean relationship for the complex as illustrated in Figure 8B:\n\nSubunit1 · Subunit2 · Subunit3 = Complex\n\nEntitySet is a class in the Reactome data model51 used to collect a set of entities with the same function in a biochemical reaction and is routinely used to collect protein isoforms. We expanded an EntitySet instance into an “OR” relationship among annotated members:\n\nMember1 + Member2 + Member3 = EntitySet\n\nThus, by converting biochemical reactions, complexes, and EntitySets into a set of Boolean relationships, we converted a Reactome pathway into a Boolean network.\n\nWe adopted the constrained fuzzy logic simulation approach31,32 to simulate the dynamic behavior of Boolean networks that are automatically converted from Reactome pathways by extending two-state Boolean variables to continuous variables with values between 0 and 1, inclusively. This allows our modeling approach to be applied to continuous ‘omics data types and drug-target assay values without the need for discretization. Based on the minimum supporting assay values for drug-target interactions loaded from the drug data sources, we calculated inhibition or activation strength by using the following hard-coded piecewise function:\n\n\n\nFunction f(input) may be a Hill function31,32 or an Identity function. For the four simulations conducted with the “Signaling by SCF-KIT” pathway, the Identity function was used.\n\nTo measure the perturbation impact caused by drug action, we developed a relative impact score based on the time step plots for individual entities in the pathway. We conducted the simulation twice: one with drugs as the perturbation, and another without drugs as the reference. The relative impact score was then calculated as follows (AUC for area under curve of fuzzy logic variable value vs. time step):\n\n\n\nOur test results indicated that the above defined relative impact score, ranging between -1.0 and 1.0, inclusively, converged within a limited number of time steps in most cases.\n\nWe adopted the three-tier software architecture: The data tier provides drug-target interactions from the Cancer Targetome and DrugCentral, contents from the Reactome database, and the Reactome FI network; The server tier fetches the content from the data tier and then send it to the front-end app via a RESTful API (application programming interface). The API also provides methods for Boolean network construction and fuzzy logic simulation. The server was implemented upon the Spring framework (https://spring.io) along with the Hibernate OR (object/relational) mapping (http://hibernate.org); the front end provides all user interfaces.\n\nOur implementation of Boolean network in Java referred to the Java implementation of open source CellNOptR52, a comprehensive Boolean network modeling toolkit for 'omics data, hosted at https://github.com/saezlab/cytocopter/tree/master. The implementation of constrained fuzzy logic simulation referred to the code (version 1.2) hosted at https://github.com/saezlab/CNORfuzzy by Saez Lab. The simulation ran until either the maximum difference between two consecutive iterations was less than 1.0 × 10-6, or the total number of iterations reached the larger of 100 or 1.2 × total number of variables in the Boolean network. To calculate the relative impact scores, we expanded the plots of fuzzy variable values vs. time steps based on reached attractors by 20 time steps each time. This expansion stopped when the maximum difference between two consecutive expansions was either less than 0.01, or the expansion reached a total of 1000 time steps. As the default setup for simulation, 1.0 was assigned as the initial values for those entities annotated as inputs of reactions forming loops or inputs that are not annotated as outputs in the pathway.\n\nWe used Java 8 (https://java.com) and Eclipse (https://www.eclipse.org) as the integrated development environment (IDE) for programming. ReactomeFIViz is released through the Cytoscape App Store (http://apps.cytoscape.org/apps/reactomefiplugin). The code is open source, hosted at GitHub (https://github.com/reactome-fi/CytoscapePlugIn).\n\n\nSoftware availability\n\nHome page for user guide describing procedures on how to use ReactomeFIViz features for drug visualization and modeling: https://reactome.org/tools/reactome-fiviz\n\nCytoscape app store: http://apps.cytoscape.org/apps/reactomefiplugin\n\nLatest source code: https://github.com/reactome-fi/CytoscapePlugIn\n\nSource code as at the time of publication: https://github.com/reactome-fi/CytoscapePlugIn/releases/tag/f1000_drug_paper\n\nArchived source code as at the time of publication: http://doi.org/10.5281/zenodo.323795553\n\nLicense: Creative Commons Attribution 4.0 International (CC BY 4.0) License (https://reactome.org/license).\n\n\nData availability\n\nThe TCGA ovarian cancer mutation data file: http://cpws.reactome.org/caBigR3WebApp2018/ov.maf.txt.zip\n\nZenodo: Reactome_FIViz_Drug_Visualization_Supp, https://doi.org/10.5281/zenodo.323944154\n\nThis project contains the following extended data:\n\nTable S1. Significant pathways for the TCGA ovarian cancer FI network. Pathways enriched by the mutated genes in the TCGA ovarian cancer FI network with FDR <= 0.05 are listed. Highlighted pathways contain EGFR (in yellow except Focal adhesion(K)), PAK3 (in brown), or TNIK (in green), as discussed in the main text. (R) indicates pathways collected from Reactome, (K) from KEGG (1), (N) from NCI Pathway Interaction Database (2), (P) from Panther Pathways.\n\n1. Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017 Jan 4;45(D1):D353–61.\n\n2. Schaefer CF, Anthony K, Krupa S, Buchoff J, Day M, Hannay T, et al. PID: the Pathway Interaction Database. Nucleic Acids Res. 2009 Jan;37(Database issue):D674–9.\n\nFigure S1. Constrained fuzzy logic simulation results for entity PI(3,4,5)P3 [plasma membrane] (https://reactome.org/PathwayBrowser/#/R-HSA-1433557&SEL=R-ALL-179838&PATH=R-HSA-162582,R-HSA-9006934) in the “Signaling by SCF-KIT” pathway from 4 simulations. A: Simulations conducted with the default setup provided by ReactomeFIVIz. B: Same as A except the initial value of PRKCA was reduced from 1.0 to 0.5. Logic fuzzy values prior to time step 11 are the same for all four simulations, which is 0.0.\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 project was supported by NIH grants 2U41HG003751 (L.S., G.W.), T15LM007088 (A.S.B.), 1R01CA192405 (S.K.M), and 5UL1TR000128 (S.K.M., G.W.).\n\nThe funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Drs. 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Reference Source\n\nVastrik I, D’Eustachio P, Schmidt E, et al.: Reactome: a knowledge base of biologic pathways and processes. Genome Biol. 2007; 8(3): R39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerfve C, Cokelaer T, Henriques D, et al.: CellNOptR: a flexible toolkit to train protein signaling networks to data using multiple logic formalisms. BMC Syst Biol. 2012; 6: 133. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu G: ReactomeFIViz_Drug_Visualization (Version 1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3237955\n\nWu G: Reactome_FIViz_Drug_Visualization_Supp. 2019. http://www.doi.org/10.5281/zenodo.3239441" }	[ { "id": "50786", "date": "15 Jul 2019", "name": "Fuhai Li", "expertise": [ "Reviewer Expertise Drug repositioning", "drug combination prediction", "computational 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 study, the authors improved a cytoscape network tool to analyze and visualize drug target interaction in the context of signaling pathways. The detailed examples are very helpful to indicate the importance of the functions of the tool. Overall it is well written. A couple of comments are as follows:\n\nDiscuss the possibility of zoom in/out the signaling pathways, considering that multiple signaling pathways are often activated in a specific disease. If it is possible to list/visualize all drugs targeting on all the pathways, and users can check the details, like the google map.\n\nFor the drug-target interaction, it is better to indicate the target names directly beside the color bar (because it is hard to match so many colors to drug names in the legend area)\n\nMinor: the resolution of Figure 1 is low. The subfigures (though these sub-figures are presented again the manuscript) are not clear.\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": "4849", "date": "22 Aug 2019", "name": "Guanming Wu", "role": "Author Response", "response": "Dear Dr. Li, Thanks a lot for reviewing our article. Please see our reply to your comments below: 1. Discuss the possibility of zoom in/out the signaling pathways, considering that multiple signaling pathways are often activated in a specific disease. If it is possible to list/visualize all drugs targeting on all the pathways, and users can check the details, like the google map. Thank you for the suggestion. ReactomeFIViz currently provides a feature to perform systems pathway analysis (Search for ‘Perform systems pathway impact analysis’ in our user guide, https://reactome.org/userguide/reactome-fiviz). Results from this analysis show all pathways potentially impacted by a drug. However, we have not provided a Google Map like visualization tool in ReactomeFIViz. But in our web site (www.reactome.org), we provide two views for all events annotated in the database: overview (https://reactome.org/PathwayBrowser/) and foamtree view (https://reactome.org/reacfoam/?species=48887&color=COPPER). We are considering porting these views in future iterations of ReactomeFIViz. 2. For the drug-target interaction, it is better to indicate the target names directly beside the color bar (because it is hard to match so many colors to drug names in the legend area) Thanks for your suggestion. Within the ReactomeFIViz app, users can mouse-over the target to see the target name displayed. We avoid to display target names in the plot to reduce clutter. 3. Minor: the resolution of Figure 1 is low. The subfigures (though these sub-figures are presented again the manuscript) are not clear. Sub-figures in Figure 1 are meant as thumbnails only, not intended to be read by readers." } ] }, { "id": "50435", "date": "19 Jul 2019", "name": "Aritro Nath", "expertise": [ "Reviewer Expertise bioinformatics", "pharmacogenomics", "cancer genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Blucher et al. present important updates to the ReactomeFIViz app for Cytoscape. They added several very useful features to this popular plugin that now enables easy visualization of drug and their targets in the context of biological pathways. In addition to retrieving and overlaying the drug-target information on the Reactome pathways, the updated app provides convenient visual representations of several layers of related information. Moreover, they added the capability to model the impact of single or multiple drugs on the pathways using a simple yet elegant approach based on Boolean Network modeling. Overall, the authors have done a great job in providing a thorough description of the new capabilities of the software.\n\nI only have some minor comments and observations:\nThe fuzzy logic modeling examples are very well described but It might be useful if the description included some discussion on the biological interpretation of these results, especially in the context of precision medicine. I found the online documentation for the reactomeFIViz documentation very useful to follow the analyses discussed in the manuscript. However, I was not able to test the “systems pathway impact analysis” which kept returning an HTTP status 500 error message. I am not sure if it’s a user-specific error, but it might be worth checking. Some of the drug-target overlays are very difficult to read because of high edge-density. It might help if the edges were semi-transparent.\n\nConsider using a color-blind friendly palette for visualizing simulation outputs, as the red-green gradient may be challenging for some users to differentiate.\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": "4850", "date": "22 Aug 2019", "name": "Guanming Wu", "role": "Author Response", "response": "Dear Dr. Nath, Thanks a lot for reviewing our paper and checking out our software. Please see our reply to your comments below: 1. The fuzzy logic modeling examples are very well described but It might be useful if the description included some discussion on the biological interpretation of these results, especially in the context of precision medicine. We believe we have addressed this point in our discussion of the modeling results (Figure 5) for the “p-STAT dimers” simulation. In particular, we discussed how this modeling framework could be used to better understand effects of patient alterations in the PRKCA gene. 2. I found the online documentation for the reactomeFIViz documentation very useful to follow the analyses discussed in the manuscript. However, I was not able to test the “systems pathway impact analysis” which kept returning an HTTP status 500 error message. I am not sure if it’s a user-specific error, but it might be worth checking. Thank you for pointing this out! There was a configuration error in the server side. This has been fixed now. 3. Some of the drug-target overlays are very difficult to read because of high edge-density. It might help if the edges were semi-transparent. Thanks for the suggestion! We will consider trying using semi-transparent edges and see if it helps in our future development. ReactomeFIViz provides a filtering feature. Users may use this feature to limit to a specific drug or drugs based on some filtering criteria. 4. Consider using a color-blind friendly palette for visualizing simulation outputs, as the red-green gradient may be challenging for some users to differentiate. Good suggestion! We will work to incorporate color-blind friendly palettes in the future." } ] }, { "id": "50787", "date": "26 Jul 2019", "name": "Tero Aittokallio", "expertise": [ "Reviewer Expertise Computational systems medicine", "Biostatistics", "Data mining" ], "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 an enhanced Cytoscape plugin, called ReactomeFIViz, which enables researchers to visualize drug-target interactions according to strength of supporting binding assay evidence, visualize drug-target interactions in the contexts of pathways and functional interaction networks, and to perform Boolean Network modeling to investigate the impact of drugs on pathways. This seems like a highly useful tool for the community, and I have only a couple of comments on how to improve the manuscript, as well as a few suggestions on potential future versions of ReactomeFIViz.\nThere are several drug-target interaction network visualization and drug repurposing analysis tools available as web-based software implementations, for instance, those reviewed in our recent article1. Please cite those that are relevant for the present work, and also describe the added value of ReactomeFIViz in the context of precision medicine applications.\n\nCancer Targetome aggregates drug-target data from various databases in different formats; for example, DrugBank simply reports binary drug-target interactions and does not provide quantitative bioactivity measurements (i.e. IC50, Kd, Ki). Similarly, IUPHAR/BPS Guide to Pharmacology reports median bioactivity values in negative log. Please describe how these data were combined?\n\nIt remains unclear how the evidence from multiple bioactivity assays (e.g. Kd, Ki, EC50, IC50) are summarized into an interaction strength for pathway analyses (as shown in Figure 2 and Table 1). This is important especially when there are multiple, perhaps contradicting evidence from multiple assays for the same drug-target pair; which assay is considered most informative in such cases?\n\nWe have initiated an open-data platform, called DrugTargetCommons (DTC), with aim to annotate bioactivity data with assay information to enable more harmonized compound-target analyses. This community-based effort involves both approved drugs and investigational compounds, and perhaps something the authors would like to consider in the future version of ReactomeFIViz.\n\nThe drug-target interaction network analyses shown in Fig. 7 are interesting, but it remains unclear whether the extracted drug-target information includes also mutant targets, or is the visualization based only on wild-type targets? Selective targeting of the mutant protein (e.g. oncogene) often leads to better efficacy and less side effects, especially if the protein is essential in healthy cells. For instance, DTC includes more than 200 mutant targets with binding affinities for hundreds of drugs.\n\nIntroduction mentions several current challenges in precision medicine, such as identifying biomarkers to guide treatment decisions, rationally designing combination therapies for improved efficacy, reducing therapeutic toxicity and side effects, and managing both intrinsic and adaptive drug resistance. The authors should summarize in Discussion which of these challenges ReactomeFIViz can currently address, and how, and which will be topics of future research.\n\nSpecific comments:\nFigure 1: the text is so small in the panels that it becomes unreadable.\n\nFigure 2: several targets are marked with same colors, can you redefine coloring based on pathways?\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": "4851", "date": "22 Aug 2019", "name": "Guanming Wu", "role": "Author Response", "response": "Dear Drs Aittokallio and Tanoli, Thanks a lot for reviewing our paper, many great comments and suggestions, and being willing to share your data with us. We are happy to talk more on how to integrate your drug data into ReactomeFIViz offline. Please see our reply to your comments below: 1. There are several drug-target interaction network visualization and drug repurposing analysis tools available as web-based software implementations, for instance, those reviewed in our recent article1. Please cite those that are relevant for the present work, and also describe the added value of ReactomeFIViz in the context of precision medicine applications. Thanks for your suggestion and referring your article to us. Our paper is focused on placing drug-target interactions in the context of Reactome pathways and FI network to show the larger biological context, whereas applications reviewed in your article are focused on drug-target networks or drug-target interaction prediction. To limit the scope of our paper, we have not addressed applications in that category. 2. Cancer Targetome aggregates drug-target data from various databases in different formats; for example, DrugBank simply reports binary drug-target interactions and does not provide quantitative bioactivity measurements (i.e. IC50, Kd, Ki). Similarly, IUPHAR/BPS Guide to Pharmacology reports median bioactivity values in negative log. Please describe how these data were combined? Correct. Cancer Targetome aggregates from several different resources and assigns evidence levels to drug-target interactions according to supporting evidence. Also, we note that in the bulk download we used for IUPHAR/BPS interactions, bioactivity values were listed in several formats, one of which was negative log and one of which was untransformed values. For more information, please see our Cancer Targetome paper (https://www.ncbi.nlm.nih.gov/pubmed/28964549). 3. It remains unclear how the evidence from multiple bioactivity assays (e.g. Kd, Ki, EC50, IC50) are summarized into an interaction strength for pathway analyses (as shown in Figure 2 and Table 1). This is important especially when there are multiple, perhaps contradicting evidence from multiple assays for the same drug-target pair; which assay is considered most informative in such cases? To clarify, in Figure 2 we show the bioactivity assay results listed separately by each assay type, so there is no summarization across assay types. For showing the drug-target evidence connected to a pathway, or when we use a threshold on binding assay value (as in Table 1), the minimum binding assay value across assay types is always used. Our app allows the user to filter drug-target data according to assay type and strength. We agree that the issue of contradicting evidence from different assays presents a challenge; in Cancer Targetome and in the ReactomeFIViz app we have elected to show all assay values aggregated and then allow the user to prioritize accordingly. 4. We have initiated an open-data platform, called DrugTargetCommons (DTC), with aim to annotate bioactivity data with assay information to enable more harmonized compound-target analyses. This community-based effort involves both approved drugs and investigational compounds, and perhaps something the authors would like to consider in the future version of ReactomeFIViz. Yes, this would be an excellent addition for future versions of ReactomeFIViz, in particular the inclusion of investigational compounds. We will be happy to discuss a possible collaboration off-line. 5. The drug-target interaction network analyses shown in Fig. 7 are interesting, but it remains unclear whether the extracted drug-target information includes also mutant targets, or is the visualization based only on wild-type targets? Selective targeting of the mutant protein (e.g. oncogene) often leads to better efficacy and less side effects, especially if the protein is essential in healthy cells. For instance, DTC includes more than 200 mutant targets with binding affinities for hundreds of drugs. Great point. Our drug-target interactions currently do not include mutation-level information. This is a current limitation of the aggregation done by Cancer Targetome and we would definitely like to include mutation level information in future iterations in the ReactomeFiViz application. 6. Introduction mentions several current challenges in precision medicine, such as identifying biomarkers to guide treatment decisions, rationally designing combination therapies for improved efficacy, reducing therapeutic toxicity and side effects, and managing both intrinsic and adaptive drug resistance. The authors should summarize in Discussion which of these challenges ReactomeFIViz can currently address, and how, and which will be topics of future research. Thank you for the suggestion. In the Discussion section, we touched topics include drug prioritization, drug combination, and drug resistance. Currently other features provided in ReactomeFIViz, which are not covered in this paper, may be able to address some other aspects of precision medicine (e.g. biomarker discovery based on gene expression. For an example, please see https://genomebiology.biomedcentral.com/articles/10.1186/gb-2012-13-12-r112). We will try to write a future paper to cover these topics in a more comprehensive way. Specific comments: · Figure 1: the text is so small in the panels that it becomes unreadable. This was also pointed out by another reviewer. The sub-figures in Figure 1 are used as thumbnails only, illustrating new features we have developed for visualizing drug-target interactions using ReactomeFIViz. They are not intended to be read. · Figure 2: several targets are marked with same colors, can you redefine coloring based on pathways? This is an interesting idea, and one we will take a look in our future development of ReactomeFIViz. However, it may be difficult to implement without confusing users since there are just too many pathways in Reactome." } ] } ]	1	https://f1000research.com/articles/8-908
https://f1000research.com/articles/7-1667/v1	19 Oct 18	{ "type": "Software Tool Article", "title": "FDTool: a Python application to mine for functional dependencies and candidate keys in tabular data", "authors": [ "Matt Buranosky", "Elmar Stellnberger", "Emily Pfaff", "David Diaz-Sanchez", "Cavin Ward-Caviness", "Elmar Stellnberger", "Emily Pfaff", "David Diaz-Sanchez", "Cavin Ward-Caviness" ], "abstract": "Functional dependencies (FDs) and candidate keys are essential for table decomposition, database normalization, and data cleansing. In this paper, we present FDTool, a command line Python application to discover minimal FDs in tabular datasets and infer equivalent attribute sets and candidate keys from them. The runtime and memory costs associated with seven published FD discovery algorithms are given with an overview of their theoretical foundations. We conclude that FD_Mine is the most efficient FD discovery algorithm when applied to datasets with many rows (> 100,000 rows) and few columns (< 14 columns). This puts it in a special position to rule mine clinical and demographic datasets, which often consist of long and narrow sets of participant records. The structure of FD Mine is described and supplemented with a formal proof of the equivalence pruning method used. FDTool is a re-implementation of FD Mine with additional features added to improve performance and automate typical processes in database architecture. The experimental results of applying FDTool to 12 datasets of different dimensions are summarized in terms of the number of FDs checked, the number of FDs found, and the time it takes for the code to terminate. We find that the number of attributes in a dataset has a much greater effect on the runtime and memory costs of FDTool than does row count. The last section explains in detail how the FDTool application can be accessed, executed, and further developed.", "keywords": [ "Functional dependencies", "Data mining", "Electronic health records", "Relational database", "FDTool", "Rule discovery" ], "content": "Introduction\n\nFunctional dependencies (FDs) are key to understanding how attributes in a database schema relate to one another. An FD defines a rule constraint between two sets of attributes in a relation1 r(U), where U = {v1,v2,…,vm} is a finite set of attributes (Yao et al., 2002). A combination of attributes over a dataset is called a candidate (Yao et al., 2002). An FD X → Y asserts that the values of candidate X uniquely determine those of candidate Y (Yao et al., 2002). For example, the social security number (SSN) attribute in a dataset of public records functionally determines the first name attribute. Because the FD holds, we write {SSN} → {first_name}.\n\nDefinition 1. A functional dependency X → Y, where X,Y ⊆ U, is satisfied by r(U), if for all pairs of tuples ti, tj ∈ r(U), we have that ti [X] = tj [X] implies ti [Y] = tj [Y] (Yao & Hamilton, 2008).\n\nIn this case, X is the left-hand side of an FD, and Y is the right-hand side (Yao et al., 2002). If Y is not functionally dependent on any proper subset of X, then X → Y is minimal (Yao et al., 2002). Minimal FDs are our only concern in rule mining FDs, since all other FDs are logically implied. For instance, if we know {SSN} → {first_name}, then we can infer that {SSN, last_name} → {first_name}.\n\nThe search space for FDs can be represented as a power set lattice of nonempty attribute combinations. Figure 1 gives the nonempty attribute combinations of a relation r(U) such that U = {A,B,C,D}. There are 2n – 1 = 24 – 1 = 15 attribute subsets in the power set lattice (Yao & Hamilton, 2008). Each combination X of the attributes in U can be the left-hand side of an FD X → Y such that X → Y is satisfied by relation r(U) (Yao & Hamilton, 2008). Since the attribute set itself U trivially determines each one of its proper subsets, it can be ignored as a candidate. There remain 2n – 2 = 24 – 2 = 14 nonempty subsets of U that are to be considered candidates.\n\nThere are n · 2n–1 – n = 4 · 24–1 – 4 = 28 edges (or arrows) in the semi-lattice of the complete search space for FDs in relation r(U) (Yao & Hamilton, 2008). The size of the search space for FDs is exponentially related to the number of attributes in U. Hence, the search space for FDs increases quite significantly when there is a greater number of attributes in U. For instance, when there are 12 attributes in a relation, the search space for FDs climbs to 24,564. This gives reason to be cautious of runtime and memory costs when deploying a rule mining algorithm to discover FDs.\n\nThe algorithms used to discover FDs differ in their approach to navigating the complete search space of a relation. Their candidate pruning methods vary and sometimes the methods used to validate FDs do as well. These differences affect runtime and memory behavior when used to process tables of different dimensions.\n\nA common data structure used to validate FDs is the partition. A partition places tuples that have the same values on an attribute into the same group (Yao et al., 2002).\n\nDefinition 2. Let X ⊆ U and let t1,…,tn be all the tuples in a relation r(U). The partition over X, denoted ∏X, is a set of the groups such that ti and tj, 1 ≤ i, j ≤ n, i 1≠ j, are in the same group if and only if ti [X] = tj [X] (Yao et al., 2002).\n\nIt follows from Definition 2 that the cardinality of the partition card(∏A(r)) is the number of groups in partition ∏A (Yao & Hamilton, 2008). The cardinality of the partition offers a quick approach to validating FDs in a dataset.\n\nTheorem 1. An FD X → Y is satisfied by a relation r(U) if and only if card(∏X) = card(∏XY) (Huhtala et al., 1999).\n\nTheorem 1 provides an efficient method to check whether an FD X → Y holds in a relation2. Huhtala et al. (1999) proved it to support a fast validation method for relations consisting of a large number of tuples.\n\nEfforts in relational database theory have lead to more runtime and memory efficient methods to check the complete search space of a relation for FDs. In place of needing each arrow in a semi-lattice checked, we can infer the FDs that logically follow from those already discovered. Such FDs are to be discovered as a consequence of Armstrong’s Axioms (Maier, 1983) and the inference axioms derivable from them (Ramakrishnan & Gehrke, 2000), which are\n\n– Reflexivity: Y ⊆ X implies X → Y;\n\n– Augmentation: X → Y implies XZ → YZ;\n\n– Transitivity: X → Y and Y → Z imply X → Z;\n\n– Union: X → Y and X → Z imply X → YZ;\n\n– Decomposition: X → YZ implies that X → Y and X → Z.\n\nThese axioms signal the distinction between FDs that can be inferred from already discovered FDs and those that cannot (Maier, 1983). Exploiting what can be derived from Armstrong’s Axioms allows us to avoid having to check many of the candidates in a search space.\n\nDefinition 3. Let F be a set of functional dependencies over a dataset D and X be a candidate over D. The closure of candidate X with respect to F, denoted X+, is defined as {Y \| X → Y can be deduced from F by Armstrong’s Axioms} (Yao & Hamilton, 2008).\n\nThe nontrivial closure3 of candidate X with respect to F is defined as X* = X+ \\ X and written X* (Yao & Hamilton, 2008). Definition 3 gives room to elegantly define keys. Informally, a key implies that a relation does not have two distinct tuples with the same values on those attributes. Keys uniquely identify all tuple records.\n\nDefinition 4. Let R be a relational schema and X be a candidate of R over a dataset D. If X ∪ X* = R, then X is a key (Yao et al., 2002).\n\nA candidate key X of a relation is a minimal key for that relation. This means that there is no proper subset of X for which Definition 4 holds.\n\n\nRule mining algorithms\n\nExisting functional dependency algorithms are split between three categories: Difference- and agree-set algorithms (e.g., Dep-Miner, FastFDs), Dependency induction algorithms (e.g., FDEP), and Lattice traversal algorithms (e.g., TANE, FUN, FD_Mine, DFD) (Papenbrock et al., 2015).\n\nDifference- and agree-set algorithms model the search space of a relation as the cross product of all tuple records (Papenbrock et al., 2015). They search for sets of attributes agreeing on the values of certain tuple pairs. Attribute sets only functionally determine other attribute sets whose tuple pairs agree, i.e., agree-sets (Asghar & Ghenai, 2015; Papenbrock et al., 2015). Then, agree-sets are used to derive all minimal FDs.\n\nDependency induction algorithms assume a base set of FDs in which each attribute functionally determines each other attribute (Papenbrock et al., 2015). While iterating through row data, observations are made that require certain FDs to be removed from the base set and others added to it. These observations are made by comparing tuple pairs based on the equality of their projections. After each record in a dataset is compared, the FDs left in the base set are considered valid, minimal and complete (Papenbrock et al., 2015).\n\nLattice traversal algorithms model the search space of a relation as a power set lattice. Most of such algorithms, (i.e., TANE, FUN, FD_Mine) use a level-wise approach to traversing the search space of a relation from the bottom-up (Papenbrock et al., 2015). They start by checking4 for FDs that are singleton sets on the left-hand side and iteratively transition to candidates of greater cardinality.\n\nPapenbrock et al. (2015) released an experimental comparison of the aforementioned FD discovery algorithms. The seven algorithms were re-implemented in Java based on their original publications and applied to 17 datasets of various dimensions. They found that none of the algorithms are suited to yield the complete result set of FDs from a dataset consisting of 100 columns and 1 million rows (Papenbrock et al., 2015). Hence, it is a matter of discretion to choose the algorithm best fitting the dimensions of a dataset.\n\nThe experimental results show that lattice traversal algorithms are the least memory efficient, since each k-level5 can be a factor greater than the size of the previous level (Papenbrock et al., 2015). Difference- and agree-set algorithms and dependency induction algorithms perform favorably in memory experiments as a result of their operating directly on data and efficiently storing result sets. Lattice traversal algorithms scale poorly on tables with many columns (≥ 14 columns) due to memory limits (Papenbrock et al., 2015).\n\nLattice traversal algorithms are the most effective on datasets with many rows, because their validation method6 operates on attribute sets as opposed to data (Papenbrock et al., 2015). This puts such algorithms in a special position to rule mine clinical and demographic record datasets, which often consist of long and narrow sets of participant records. Difference- and agree-set algorithms and dependency induction algorithms commonly reach time limits when applied to datasets of these dimensions (> 100,000 rows) (Papenbrock et al., 2015).\n\nLattice traversal algorithms iterate through k-levels represented in a power set lattice. If the lattice is traversed from the bottom-up, we say the algorithm is level-wise.\n\nDefinition 5. Let X1, X2,…, Xk, Xk+1 be (k + 1) attributes over a database D. If X1X2… Xk → Xk+1 is an FD with k attributes on its left hand side, then it is called a k-level FD (Yao et al., 2002).\n\nThe search space for FDs is reduced at the end of each iteration using pruning rules. Pruning rules check the validity of candidates not yet checked with FDs already discovered and those inferred from Armstrong’s Axioms (Yao & Hamilton, 2008). After a search space is pruned, an Apriori_Gen principle generates k-level candidates with the (k – 1)-level candidates that were not pruned (Yao & Hamilton, 2008).\n\nApriori_Gen:\n\n– oneUp: generates all possible candidates in Ck from those in Ck–1.\n\n– oneDown: generates all possible candidates in Ck–1 from those in Ck.\n\nLevel-wise lattice traversal algorithms stop iterating after all candidates in a search space are pruned. In this case, Apriori_Gen generates the null set ∅ raising a flag for the algorithm to terminate. This has the effect of shortening runtime to the degree that FDs are discovered and others are inferred.\n\nThe level-wise lattice traversal algorithms TANE, FUN, and FD_Mine differ in terms of pruning rules. FUN and FD_Mine expand on the pruning rules of TANE. Released by Huhtala et al. (1999), TANE prunes a search space on the basis that only minimal and non-trivial7 FDs need be checked. TANE restricts the right-hand side candidates C+ for each attribute combination X to the set\n\n\n\nwhich contains all the attributes that the set X may still functionally determine (Papenbrock et al., 2015). The set C+ is used in the following pruning rules (Papenbrock et al., 2015).\n\n• Minimality pruning: If an FD X \\ A → A holds, A and all B ∈ C+ (X) \\ X can be removed from C+ (X).\n\n• Right-hand side pruning: If C+ (X) = ∅, the attribute combination X can be pruned from the lattice, as there are no more right-hand side candidates for a minimal FD.\n\n• Key pruning: If the attribute combination X is a key, it can be pruned from the lattice.\n\nKey pruning implies that all supersets of a key, i.e., super keys, can be removed, since they are by definition non-minimal (Huhtala et al., 1999).\n\n\nFD_Mine\n\nLike TANE and FUN, FD_Mine is structured around the level-wise lattice traversal approach and the aforemented pruning rules. Unlike the other two algorithms, FD_Mine, authored by Yao et al. (2002), uses the concept of equivalence as means to more exhaustively prune the search space of a candidate (Papenbrock et al., 2015). Informally, attribute sets are equivalent if and only if they are functionally dependent on each other (Papenbrock et al., 2015).\n\nThe proofs demonstrating that no useful information is lost in pruning candidates from equivalent attribute sets are reproduced in this section and were originally developed by Yao & Hamilton (2008). The equivalence pruning method can be derived directly from Armstrong’s Axioms.\n\nDefinition 6. Let X and Y be candidates over a dataset D. If X → Y and Y → X hold, then we say that X and Y are an equivalence and denote it as X ↔ Y.\n\nAfter a k-level is fully validated, i.e., each k-level candidate is checked, FD_Mine determines equivalent attribute sets with the FDs already discovered.\n\nTheorem 2. Let X, Y ⊆ U. If Y ⊆ X+ and X ⊆ Y+, then X ↔ Y (Yao & Hamilton, 2008).\n\nProof. Since X → X+ and Y ⊆ X+, Decomposition implies that X → Y. By a similar argument, Y → X holds. Because X → Y and Y → X, we have by definition that X ↔ Y holds.\n\nLemma 3 and Lemma 4 are derived from Armstrong’s Axioms with the assumption of the equivalence X ↔ Y.\n\nLemma 3. Let W,X,Y,Y',Z ⊆ U and Y ⊆ Y'. If X ↔ Y and XW → Z, then Y'W → Z (Yao & Hamilton, 2008).\n\nProof. Suppose that X ↔ Y and XW → Z. This implies that X → Y. By Augmentation, YW → XW. By Transitivity, YW → XW and XW → Z give that YW → Z. By Augmentation, Y' \\ Y can be added to both sides of YW → Z to give that YW(Y' \\ Y) → Z(Y' \\ Y). By Y ⊂ Y', we know that Y'W → Z(Y' \\ Y). Then, by Decomposition, Y'W → Z.\n\nLemma 4. Let W,X,Y,Z ⊆ U. If X ↔ Y and WZ → X, then WZ → Y (Yao & Hamilton, 2008).\n\nProof. By X ↔ Y, we know that X → Y. By Transitivity, WZ → X and X → Y imply WZ → Y.\n\nTheorem 2 checks attribute sets X and Y for the equivalence X ↔ Y. FD_Mine assumes that the attribute set Y is generated before X. By Lemma 3 and Lemma 4, we know that for equivalence X ↔ Y, no further attribute sets Z such that Y ⊆ Z need be checked (Yao & Hamilton, 2008). Hence, Y is deleted as a result of the following pruning rule.\n\n• Equivalence pruning: If X ↔ Y is satisfied by relation r(U), then candidate Y can be deleted. (Yao & Hamilton, 2008).\n\nExploiting the equivalence pruning method leaves FD_Mine in a more aggressive position to prune candidates than TANE. This offers an advantage in terms of runtime and memory behavior (Yao et al., 2002).\n\nThe pseudo-code proposed in the second version of FD_Mine (Yao & Hamilton, 2008) will under certain circumstances output non-minimal FDs (Papenbrock et al., 2015). FD_Mine references an Apriori_Gen method (Agrawal et al., 1996) stating that for each pair of candidates p, q ∈ Ck–1 the set p ∪ q is to be placed in Ck if card(p ∪ q) = k. Example 1 shows that the Apriori_Gen method referenced and utilized by FD_Mine can violate minimality pruning by checking supersets that need not be checked. Figure 2 gives the power set lattice of the relation described in Example 1 pruned by FD_Mine.\n\nFD_Mine deletes the candidates ABC, ABE, and ABD (red ovals) as a result of finding the candidate key AB (blue hexagon). It generates supersets of AB (yellow rectangles) at the next level.\n\nExample 1. Let r(U) be a relation such that U = {A,B,C,D,E}. Suppose that AB is a key and that there are no other FDs in r(U). Since AB is a key, we know by definition that AB ∪ AB* = U. Provided this and that there are no other FDs in r(U), the candidates ABC, ABD and ABE are deleted from C3, and so C3 = Prune(Apriori_Gen(C2)) = {ACE, BCE, ACD, BCD, ADE, CDE, BDE}8. Then, C4 = {ABCD, ABCE, ACDE, ABDE, BCDE}. Because it must be that AB* = {C, D, E}, the algorithm validates the FDs ABCD → E, ABCE → D, and ABDE → C. Since E, for example, is functionally dependent on the proper subset AB ⊆ ABCD, ABCD → E is non-minimal.\n\nThe Apriori_Gen principle presented in TANE (Huhtala et al., 1999) more effectively generates candidate level Ck+1 from Ck. It requires that Ck+1 only contains the attribute sets of size k + 1 which have all their subsets of size k in Ck (Huhtala et al., 1999); i.e.,\n\n\n\nIn reference to Example 1, this method does not insert the candidate ABCD in C4, without loss of generality, because ABC ⊆ ABCD but ABC ∉ C3. Thus, the non-minimal FD ABCD → E is not checked.\n\nPrune(Ck, E, Closure)9–11\n\n01for eachS∈Ck:02for eachX∈oneDown[Ck]:03if(X⊂S)then:04if(X∈{Z\|ϒ↔Z∈E})then:#Pruning rule 105delete Sfrom Ck06ifS⊂X+then:#Pruning rule 207delete Sfrom Ck08S+=S+∪X#Pruning rule 309ifU==S+then:#Pruning rule 410delete Sfrom Ck11returnCk,Closure;\n\nFD_Mine will under the circumstance described in Example 1 set closure values incorrectly. In line 2, FD_Mine iterates through Ck–1, as opposed to oneDown [Ck], which can cause the Prune() function to ignore setting the closure values of certain candidates. In Example 1, FD_Mine does not accurately set the closure ABCD to E, since E is not saved to the closure values of the candidates ACD, BCD ⊆ ABCD at the previous level. Iterating through oneDown [Ck] sets the closure of a candidate to the union of the closure values of its proper subsets, so that the closure values of deleted candidates are not lost among their supersets.\n\nProperly assigned closure values can allow the algorithm to avoid checking many non-minimal FDs. This is because the ObtainFDs module, i.e., the validation method, only checks12 the right-hand side attributes vi for which vi ∈ U \\ X+ (Yao & Hamilton, 2008). Hence, provided that Pruning rule 3 asserts the equality ABCD* = E, ABCD → E need not be checked.\n\n\nOperation\n\nFDTool (Buranosky, 2018) is a command line Python application executed with the following statement: $ fdtool /path/to/file13. For Windows users, this is to be run from the directory in which the executable fdtool.exe resides, which will likely be C:\\Python27\\Scripts for those installing with pip install fdtool. For other systems, installation automatically inserts the file path to the fdtool command in the PATH variable. /path/to/file is the absolute or relative path to a .txt, .csv, or .pkl file containing a tabular dataset. If the data file has the extension .txt or .csv, FDTool detects the following separators: comma (‘,’), bar (‘\|’), semicolon (‘;’), colon (‘:’), and tilde (‘∼’). The data is read in as a Pandas data frame14.\n\nDependencies:\n\n1. Python2 (https://www.python.org/), recommended version 2.7.8 or later.\n\n2. Pandas data analysis library (https://pandas.pydata.org/) via: pip install pandas.\n\nFDTool provides the user with the minimal FDs, equivalent attribute sets and candidate keys mined from a dataset. This is given with the time (s) it takes for the code to terminate (after reading in data), the row count and attribute count of the data, the number of FDs and equivalent attribute sets found, and the number of FDs checked. This is printed on the terminal after the code is executed as shown in Figure 3. The information is saved to a .FD_Info.txt file.\n\nFigure 3 shows the printed output of FDTool.exe applied to the contents of Table 1. The output file Table1. FD_Info.txt is saved to the directory from which the executable is run.\n\n\nImplementation\n\nFDTool is a Python based re-implementation of the FD_Mine algorithm with additional features added to automate typical processes in database architecture. FD_Mine was published in two papers with more detail given to the scientific concepts used in algorithms of its kind (Yao et al., 2002; Yao & Hamilton, 2008). The two versions of FD_Mine were released with different structures but make use of the same theoretical foundation (Papenbrock et al., 2015), which is fully supported in mathematical proofs of the pruning rules used (Yao & Hamilton, 2008). FDTool was coded15 with special attention given to the pseudo-code presented in the second version of FD_Mine (Yao & Hamilton, 2008).\n\nThe Python script dbschema.py in FDTool/fdtool/modules/dbschema is taken from dbschemacmd (https://www.elstel.org/database/dbschemacmd.html.en): a tool for database schema normalization working on functional dependencies (Elmasri & Navathe, 2011). It is used to take sets of FDs and infer candidate keys from them. The operation first assigns the left-hand side attribute combinations of a set of FDs to dictionary keys and their closures to the corresponding values. It then reduces the set of FDs to a minimum coverage16. Candidate keys are assembled using the minimum coverage and closure structure by adding attributes to key candidates until each minimal attribute set X for which X+ = U is found. Details on the dbschema operations are described in FDTool/fdtool/modules/dbschema/Docs.\n\n\nUse cases\n\nFDTool was initially created to help decompose datasets of medical records as part of Clinical Archived Records research for Environmental Studies (CARES). CARES currently contains 12 datasets obtained from the medical software firms Epic and Legacy. The attribute count in this database ranges from 4 to 18; the row count ranges from 42,369 to 8,201,636.\n\nTo limit the strain on computational resources, FDTool has a built in time limit of 4 hours. FDTool reaches this preset limit (triggering program termination) when applied to the PatientDemographics dataset (42369 rows × 18 columns) and the EpicVitals_TobaccoAlcOnly dataset (896962 rows × 18 columns). The remaining 10 CARES datasets are given in Table 217.\n\nThe results from Table 2 show that runtime is primarily determined by the number of attributes in a dataset. For instance, the LegacyPayors dataset (1465233 rows × 4 columns) has slightly more rows (13% increase) but far fewer attributes (60% decrease) as compared to the AllLabs dataset (1294106 rows × 10 columns). The runtime of LegacyPayers (9.4 s.) is much less than that of AllLabs (999.8 s.), because AllLabs has many more arrows in its powerset lattice,\n\n\n\nthan does LegacyPayers (28). Hence, FDTool has more FDs to check when applied to AllLabs. It is clear that the attribute count of a dataset has a much greater effect on the runtime of FDTool than does row count.\n\nMany of the arrows in the powerset lattice of a candidate are pruned by FDTool. AllLabs has 5110 arrows in its powerset lattice. However, FDTool only checks 818 FDs, as there are many inferred from the 43 FDs found. This follows from the Prune() function, which deletes many of the candidates to check partially as a result of mining 4 equivalent attribute sets. FDTool terminates after 5 k-levels when applied to AllLabs.\n\n\nFuture development\n\nWe want to improve its performance so that FDTool is better equipped to handle datasets of different dimensions. Using the dependency induction algorithm FDEP, the reach of FDTool could be extended to datasets with fewer rows and more than 100 columns (Papenbrock et al., 2015). This might also require upgrading the source code with multicore processing methods, such as a Java API, to reduce runtime and avoid reaching memory limits. A formal proof of the the dbschema operations is also desired.\n\nAnother goal is to increase the functionality provided by FDTool. This would mean implementing the pen and paper methods typically used to normalize relational schema and decompose tables. Our intent is to incorporate these changes in newer versions of FDTool, released at regular periods, so as to develop it as Python software that could automate much of what is done in the database design process.\n\n\nData availability\n\nWhile the authors fully support the open dissemination of data for verification and replication purposes, CARES data cannot be released as it contains Protected Health Information. However, the CARES data is accessible to any researcher who has an approved IRB and submits a data request to the Carolina Data Warehouse for Health (https://tracs.unc.edu/index.php/services/informatics-and-data-science/cdwh; use the “Submit a CDW-H Project Data Request” link). The authors will be happy to send the CARES data exactly as utilized in this manuscript to any researcher with an approved request from the Carolina Data Warehouse for Health.\n\n\nSoftware availability\n\nFDTool is available from the Python Package Index: https://pypi.org/project/fdtool/\n\nLatest source code: https://github.com/USEPA/FDTool.git\n\nSource code at time of publication: https://zenodo.org/record/1442843\n\nLicense: CC0 1.0 Universal. Module FDTool/fdtool/modules/dbschema released under a modified C-FSL license.", "appendix": "Author contributions\n\n\n\nMB and ES designed and implemented the software. MB wrote the manuscript. CWC supervised MB, and reviewed the manuscript. EP maintained the research data. DDS coordinated the funding for the project. All authors agreed to the final content of the manuscript.\n\n\nGrant information\n\nThis work was funded by the US Environmental Protection Agency. The work presented here does not necessarily reflect the views or policy of the EPA. Any mention of trade names does not constitute endorsement by the EPA.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nFootnotes\n\n1Each attribute vi has a finite domain, written dom(vi), representing the values that vi can take on. For a subset X = {vi,…,vj} of U, we write dom(X) for the Cartesian product of the domains of the individual attributes in X, namely, dom(X) = dom(vi) × … × dom(vj) (Yao & Hamilton, 2008). A relation r on U, denoted r(U), is a finite set of mappings {t1,…,tn} from U to dom(U) with the restriction that for each mapping t ∈ r(U), t[vi] must be in dom(vi), 1 ≤ i ≤ m, where t[vi] denotes the value obtained by restricting the mapping t to vi. Each mapping t is called a tuple and t(vi) is called the vi-value of t (Maier, 1983).\n\n2FDTool uses Theorem 1 as means to check FDs in the GetFDs module with the pandas data analysis library functions nunique() and dropduplicates.count().\n\n3FDTool saves the closure of candidates at each level before releasing it from memory at levels that follow.\n\n4 We say that an FD is checked when Theorem 1 is used to see if it holds or not (Yao et al., 2002).\n\n5Definition 5.\n\n6Theorem 1.\n\n7An FD X → A is non-trivial if and only if X ∉ A (Huhtala et al., 1999).\n\n8Since E = ∅ in this example, we can ignore the argument E in the function Prune(). For simplicity’s sake, we ignore the argument Closure.\n\n9Closure = {X+ \| X ∈ Ck V X ∈ oneDown [Ck]}.\n\n10Equivalent candidates are stored in E.\n\n11All candidates at level k are stored in Ck.\n\n12 Assume the left-hand side attribute set X.\n\n13Edit FDTool/fdtool/config.py prior to building setup with python setup.py install to change preset time limit or max k-level.\n\n14The data is read in with the Pandas function read_csv(), which is subject to the usual spacing errors associated with reading in delimiter-separated values.\n\n15FDTool was tested regularly throughout the implementation process so as to accomodate to changes made to improve runtime and memory behavior.\n\n16A set of FDs F is a coverage of another set of FDs G if every FD in G can be inferred from F; i.e., G+ ⊆ F+ (Soule, 2014). F is a minimum coverage of G if F is the smallest set of FDs that covers G (Soule, 2014).\n\n17OS: Windows 10; Installed memory (RAM): 256 GB; Processor: Intel Core, 1 CPU; Clock speed: 2.19 GHz; Python: 2.7.12; Pandas: 0.18.1.\n\n\nReferences\n\nAgrawal R, Mannila H, Srikant R, et al.: Fast discovery of association rules. Advances in knowledge discovery and data mining. 1996; 12(1): 307–328. Reference Source\n\nAsghar N, Ghenai A: Automatic discovery of functional dependencies and conditional functional dependencies: A comparative study. 2015. Reference Source\n\nBuranosky M: USEPA/FDTool: FDTool (Version v0.1.7). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1442843\n\nElmasri R, Navathe SB: Database Systems: Models, Languages, Design, and Application Programming. Pearson. 2011. Reference Source\n\nHuhtala Y, Kärkkäinen J, Porkka P, et al.: Tane: An efficient algorithm for discovering functional and approximate dependencies. Comput J. 1999; 42(2): 100–111. Publisher Full Text\n\nMaier D: Theory of Relational Databases. Computer Science Pr. 1983. Reference Source\n\nPapenbrock T, Ehrlich J, Marten J, et al.: Functional dependency discovery: An experimental evaluation of seven algorithms. Proc VLDB Endow. 2015; 8(10): 1082–1093. Publisher Full Text\n\nRamakrishnan R, Gehrke J: Database Management Systems. McGraw-Hill, Inc., New York, NY, USA, 2nd edition, 2000. Reference Source\n\nSoule R: Functional dependencies and finding a minimal cover. 2014. Reference Source\n\nYao H, Hamilton HJ: Mining functional dependencies from data. Data Min Knowl Discov. 2008; 16(2): 197–219. Publisher Full Text\n\nYao H, Hamilton H, Butz C: Fd_mine: Discovering functional dependencies in a database using equivalences. 2002. Publisher Full Text" }	[ { "id": "39685", "date": "10 Dec 2018", "name": "Howard J. Hamilton", "expertise": [ "Reviewer Expertise knowledge discovery", "data mining", "machine learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well-structured and well-written. It contains nice presentations of appropriate definitions, theorems, algorithms, examples, and use-cases.\n\nThe article has clear contributions:\nIn the theory part: It enhances the FD_Mine algorithm by improving performance and automating typical processes.\nIn the implementation part: The authors re-implement the FD_Mine algorithm, which is otherwise not publicly available as a software tool.\nIn the experiment part: The authors apply FDTool to 12 datasets of different dimensions.\nFindings: The effect of the attributes is greater than the records on the runtime and memory costs of the FDTool.\n\nAdditional contributions:\nThe article clearly describes the features of the FDTool, such as its usage and execution. It also depicts future research opportunities with respect to the further development of the FDTool.\n\nMajor Comment:\nIn the abstract, it says, “We conclude that FD_Mine is the most efficient FD discovery algorithm when applied to datasets with many rows (> 100,000 rows) and few columns (< 14 columns).” The word “conclude” does not seem appropriate here. If this result indeed follows from your research, please explain how the results shown in Table 2 support this claim with respect to all datasets shown in the table [This explanation could be added in the experimental results or experimental summary section]. However, if the conclusion is in fact being taken from Papenbrock, then wording might be adjusted to “Previous research established that FD_Mine ….” You may want to state your conclusions about your software tool.\n\nMinor corrections:\nPlease use either \"FD_Mine\" or \"FD Mine\" everywhere. The original paper had FD_Mine.\nPlease use same format (comma or no comma) for large numbers [e.g. “the row count ranges from 42,369 to 8,201,636” in the use cases section versus “AllLabs dataset (1294106” and “AllLabs has 5110 arrows in its powerset lattice” in the experimental summary]. Also please check the experimental results section.\nIn Definition 2, please correct “i 1≠ j”, presumably to “i ≠ j”.\nIn the text immediately after Definition 6, please change “determines equivalent attribute sets with” to “determines attribute sets equivalent to”.\nIn the first line of the closure section, please change “lead” to “led”.\nIn the future development section, please remove the extra \"the\" from \"A formal proof of the the dbschema operations is also desired\".\nTable 1 has no title. Please give a title for Table 1.\nTable 2 shows 11 datasets but the table title [and the description in the experimental results section] mentions 10 datasets. Could you please correct this inconsistency.\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": "46574", "date": "29 Apr 2019", "name": "Sayan Mukherjee", "expertise": [ "Reviewer Expertise Statistical and computation methods.a" ], "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 outlines in detail FDTool: a Python application to mine for functional dependencies. The paper is about developing a database tool that improves FD Mine a previous tool for functional dependencies\nThe definitions, problem statement and explanations in this paper are well written and clear. The authors also provide a good comparison of several functional dependence discovery algorithms.\n\nIn my opinion, it would be nice to have the experimental results and summary in some more detail. Also since the CARES data is not public it would be nice to have some results on available 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? 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/7-1667
https://f1000research.com/articles/8-886/v1	18 Jun 19	{ "type": "Software Tool Article", "title": "A step-by-step guide to analyzing CAGE data using R/Bioconductor", "authors": [ "Malte Thodberg", "Albin Sandelin", "Albin Sandelin" ], "abstract": "Cap Analysis of Gene Expression (CAGE) is one of the most popular 5'-end sequencing methods. In a single experiment, CAGE can be used to locate and quantify the expression of both Transcription Start Sites (TSSs) and enhancers. This is workflow is a case study on how to use the CAGEfightR package to orchestrate analysis of CAGE data within the Bioconductor project. This workflow starts from BigWig-files and covers both basic CAGE analyses such as identifying, quantifying and annotating TSSs and enhancers, advanced analysis such as finding interacting TSS-enhancer pairs and enhancer clusters, to differential expression analysis and alternative TSS usage. R-code, discussion and references are intertwined to help provide guidelines for future CAGE studies of the same kind.", "keywords": [ "CAGE", "TSS", "Enhancer", "Promoter", "DE", "Motifs" ], "content": "Background\n\nTranscriptional regulation is one of the most important aspects of gene expression. Transcription Start Sites (TSSs) are focal points of this process: The TSS act as an integration point for a wide range of molecular cues from surrounding genomic areas to determine transcription and ultimately expression levels. These include proximal factors such as chromatin accessibility, chromatin modification, DNA methylation and transcription factor binding, and distal factors such as enhancer activity and chromatin confirmation1–4.\n\nCap Analysis of Gene Expression (CAGE) has emerged as one of the dominant high-throughput assays for studying TSSs5. CAGE is based on “cap trapping”: capturing capped full-length RNAs and sequencing only the first 20–30 nucleotides from the 5’-end, so-called CAGE tags6. When mapped to a reference genome, the 5’-ends of CAGE tag identify the actual TSS for respective RNA with basepair-level accuracy. Basepair-accurate TSSs identified this way are referred to as CAGE Transcription Start Sites (CTSSs). RNA polymerase rarely initiates from just a single nucleotide: this is manifested in CAGE data by the fact that CTSSs are mostly found in tightly spaced groups on the same strand. The majority of CAGE studies have merged such CTSSs into genomic blocks typically referred to as Tag Clusters (TCs), using a variety of clustering methods (see below). TCs are often interpreted as TSSs in the more general sense, given that most genes have many CTSSs, but only a few TCs that represent a few major transcripts with highly similar CTSSs7,8. Since the number of mapped CAGE tags in a given TC is indicative of the number of RNAs from that region, the number of CAGE tags falling in given TC can be seen as a measure of expression9.\n\nAs CAGE tags can be mapped to a reference genome without the need for transcript annotations, it can detect TSSs of known mRNAs, but also mRNA from novel alternative TSSs (that might be condition or tissue dependent)7,10. Since CAGE captures all capped RNAs, it can also identify long non-coding RNA (lincRNA)11 and enhancers RNA (eRNA). It was previously shown that active enhancers are characterized by balanced bidirectional transcription, making it possible to predict enhancer regions and quantify their expression levels from CAGE data alone12,13. Thus, CAGE data can predict the locations and activity of mRNAs, lincRNAs and enhancers in a single assay, providing a comprehensive view of transcriptional regulation across both inter- and intragenic regions.\n\nBioconductor contains a vast collection of tools for analyzing transcriptomics datasets, in particular the widely used RNA-Seq and microarray assays14. Only a few packages are dedicated to analyzing 5’-end data in general and CAGE data in particular: TSRchitect15, icetea16, CAGEr17 and CAGEfightR18, see Table 1.\n\nCAGEr was the first package solely dedicated to the analysis of CAGE data and was recently updated to more closely adhere to Bioconductor S4-class standards. CAGEr takes as input aligned reads in the form of BAM-files and can identify, quantify, characterize and annotate TSSs. TSSs are found in individual samples using either simple clustering of CTSSs (greedy or distance-based clustering) or the more advanced density-based paraclu clustering method19, and can be aggregated across samples to a set of consensus clusters. Several specialized routines for CAGE data is available, such as power law normalization of CTSS counts and fine-grained TSS shifts. Finally, CAGEr offers easy interface to the large collection of CAGE data from the FANTOM consortium10. TSRchitect and icetea are two more recent additions to Bioconductor. While being less comprehensive, they aim to be more general and handle more types of 5’-end methods that are conceptually similar to CAGE (RAMPAGE, PEAT, PRO-Cap, etc.5). Both packages can identify, quantify and annotate TSSs, with TSRchitect using an X-means algorithm and icetea using a sliding window approach. icetea offers the unique feature of mapping reads to a reference genome by interfacing with Rsubread. Both CAGEr, TSRchictet and icetea offers built-in capabilities for differential expression (DE) analysis via the popular DESeq2 or edgeR packages20,21.\n\nCAGEfightR is a recent addition to Bioconductor focused on analyzing CAGE data, but applicable to most 5’-end data. It aims to be general and flexible to allow for easy interfacing with the wealth of other Bioconductor packages. CAGEfightR takes CTSSs stored in BigWig-files as input and uses only standard Bioconductor S4-classes (GenomicRanges, SummarizedExperiment, InteractionSet22,23) making it easy for users to learn and combine CAGEfightR with functions from other Bioconductor packages (e.g. instead of providing custom wrappers around other packages such as differential expression analysis). In addition to TSS analysis, CAGEfightR is the only package on Bioconductor to also offer functions for enhancer analysis based on CAGE (and similarly scoped) data. This includes enhancer identification and quantification, linking enhancers to TSSs via correlation of expression and finding enhancer clusters, often referred to as stretch- or super enhancers.\n\nIn this workflow, we illustrate how the CAGEfightR package can be used to orchestrate an end-to-end analysis of CAGE data by making it easy to interface with a wide range of different Bioconductor packages. Highlights include TSS and enhancer candidate identification, differential expression, alternative TSS usage, enrichment of motifs, GO/KEGG terms and calculating TSS-enhancer correlations.\n\n\nMethods\n\nThis workflow uses data from “Identification of Gene Transcription Start Sites and Enhancers Responding to Pulmonary Carbon Nanotube Exposure in Vivo” by Bornholdt et al24. This study uses mouse as a model system to investigate how nanotubes affect lung tissue when inhaled. Inhaled nanotubes were previously found to produce a similar response to asbestos, potentially triggering an inflammatory response in the lung tissue leading to drastic changes in gene expression.\n\nThe dataset consists of CAGE data from mouse lung biopsies: 5 mice whose lungs were instilled with water (Ctrl) and 6 mice wholes lungs were instilled with nanotubes (Nano), with CTSSs for each sample stored in BigWig-files, shown in Table 2:\n\nThe data is acquired via the nanotubes data package:\n\n\n\nThis workflow uses a large number of R-packages: Bioconductor packages are primarily used for data analysis while packages from the tidyverse are used to wrangle and plot the results. All these packages are loaded prior to beginning the workflow:\n\n\n\nWe also set some script-wide settings for later convenience:\n\n\n\nThe workflow is divided into 3 parts covering different parts of a typical CAGE data analysis:\n\n1. Shows how to use CAGEfightR to import CTSSs and find and quantify TSS and enhancer candidates.\n\n2. Shows examples of how to perform genomic analyses of CAGE dusters using core Bioconductor packages such as GenomicRanges and Biostrings. This part covers typical analyses made from CAGE data, from summarizing cluster annotation, TSS shapes and core promoter sequence analysis to more advanced spatial analyses (finding TSS-enhancer correlation links and clusters of enhancers).\n\n3. Shows how CAGEfightR can be used to prepare data for differential expression analysis with popular R packages, including DESeq2, limma and edgeR20,21,25. Borrowing from RNA-Seq terminology, differential expression can be assessed at multiple different levels: Tag cluster- and enhancer-level, gene-level and differential TSS usage26. Once differential expression results have been obtained, they can be combined with other sources of information such as motifs from JASPAR27 and GO/KEGG terms28,29,30.\n\nCAGEfightR starts analysis from CTSSs, which is the number of CAGE tag 5’-ends mapping to each basepair (bp) in the genome. CTSSs are normally stored as either BED-files or BigWig-files. CAGEfightR works on BigWig-files, since these can be efficiently imported and allow for random access.\n\nBefore starting the analysis, we recommend gathering all information (Filenames, groups, batches, preparation data, etc.) about the samples to be analyzed in a single data.frame, sometimes called the design matrix. CAGEfightR can keep track of the design matrix throughout the analysis:\n\n\n\nImporting CTSSs. We need to tell CAGEfightR where to find the BigWig-files containing CTSSs on the hard drive. Normally, one would supply the paths to each file (e.g. /CAGEdata/BigWigFiles/Sample1_plus.bw), but here we will use data from the nanotubes data package:\n\n\n\nThe first step is quantifying CTSS usage across all samples. This is often one of the most time consuming step in a CAGEfightR analysis, but it can be speed up by using multiple cores (if available, see Materials and Methods). We also need to specify the genome, which we can get from the BSgenome.Mmusculus.UCSC.mm9 genome package:\n\n\n\nThe circa 9 million CTSSs are stored as RangedSummarizedExperiment, which is the standard representation of high-throughput experiments in Bioconductor. We can inspect both the ranges and the CTSS counts:\n\n\n\nUnidirectional and bidirectional clustering for finding TSS and enhancer candidates. CAGEfightR finds clusters by calculating the pooled CTSS signal across all samples: We first normalize CTSSs count in each sample to Tags-Per-Million (TPM) values, and them sum TPM values across samples:\n\n\n\nThis will add several new pieces of information to CTSSs: The total number of tags in each library, a new assay called TPM, and the pooled signal for each CTSS.\n\nWe can use unidirectional clustering to locate unidirectional clusters, often simply called Tag Clusters (TCs), which are candidates for TSSs. The quickTSSs will both locate and quantify TCs in a single function call:\n\n\n\nNote: quickTSSs runs CAGEfightR with default settings. If you have larger or more noisy datasets you most likely want to do a more robust analysis with different settings. See the CAGEfightR vignette for more information.\n\nMany of the identified TCs will only be very lowly expressed. To obtain likely biologically relevant TSSs, we keep only TSSs expressed at more than 1 TPM in at least 5 samples (5 samples being the size of the smallest experimental group):\n\n\n\nThis removed a large number of very lowly expressed TCs, leaving us with almost 30.000 TSSs candidates for analysis.\n\nThen we turn to bidirectional clustering for identifying bidirectional clusters (BCs), which are candidate for enhancers. Similarly, we can use quickEnhancers to locate and quantify BCs:\n\n\n\nNote: quickEnhancers runs CAGEfightR with default settings. If you have larger or more noisy datasets you most likely want to do a more robust analysis with different settings. See the CAGEfightR vignette for more information.\n\nAgain, we are not usually interested in very lowly expressed BCs. As they are overall lowly expressed, we will simply filter out BCs without at least 1 count in 5 samples:\n\n\n\nAnnotating clusters with transcript models. After having located unidirectional and bidirectional clusters, we can annotate them according to known transcript and gene models. These types of annotation are store via TxDb-objects in Bioconductor. Here we will simply use UCSC transcripts included in the TxDb.Mmusculus.UCSC.mm9.knownGene package, but the CAGEfightR vignette includes examples of how to obtain a TxDb object from other sources (GFF/GTF files, AnnotationHub, etc.).\n\nStarting with the TSS candidates, we can not only annotate a TSS with overlapping transcripts, but also in what part of a transcript a TSS lies by using a hierarchical annotation scheme. As some TSSs might be very wide, we only use the TSS peak for annotation purposes:\n\n\n\nAlmost half of TSSs were found at annotated promoters, while the other half is novel compared to the UCSC known transcripts.\n\nTranscript annotation is particularly useful for enhancer candidates, as bidirectional clustering might also detect bidirectional promoters. Therefore, a commonly used filtering approached is to only consider BCs in intergenic or intronic regions as enhancer candidates:\n\n\n\nThis leaves almost 10000 enhancer candidates for analysis.\n\nMerging into a single dataset. For many downstream analyses, in particular normalization and differential expression, it is useful to combine both TSS and enhancers candidates into a single dataset. This ensures that TSSs and enhancers do not overlap, so each CAGE tag is only counted once.\n\nWe must first ensure that the enhancer and TSS candidates have the same information attached to them, since CAGEfightR will only allow merging of clusters if they have the same sample and cluster information:\n\n\n\nThen the clusters can be merged: As enhancers are the most complicated type, we keep only enhancers if a TSS and enhancer overlaps:\n\n\n\nWe finally calculate the total number of tags and TPM-scaled counts for the final merged dataset:\n\n\n\nGenome-browser figures of TSSs and enhancers. First we can simply plot some examples of TSSs and enhancers in a genome browser style figure using the Gviz package31. It takes a bit of code to setup, but the resulting tracks can be reused for later examples:\n\n\n\nA good general strategy for quickly generating genome browser plots is to first define a region of interest, and then only plotting data within that region using subsetByOverlaps. The following code demonstrates this using the first TSS:\n\n\n\nThe top track shows the pooled CTSS signal and the middle track shows the identified TC with the thick bar indicating the TSS peak (the overall most used CTSSs within the TC). The bottom track shows the known transcript model at this genomic location. In this case, the CAGE-defined TSS corresponds well to the annotation.\n\nWe can also plot the first enhancer:\n\n\n\nHere we see the bidirectional pattern characteristic of active enhancers. The bidirectional cluster is seen in the middle track, with the midpoint in thick marking the maximally balanced point within the bidirectional cluster.\n\nLocation and expression of TSSs and enhancers. In addition to looking at single examples of TSSs and enhancers, we also want to get an overview of the number and expression of clusters in relation to transcript annotation. First we extract all of the necessary data from the RangedSummarizedExperiment into an ordinary data.frame:\n\n\n\nThen we use ggplot2 to plot the number and expression levels of clusters in each annotation category:\n\n\n\n\n\nWe find that TSSs at annotated promoters are generally highly expressed. Most novel TSSs are expresse d at lower levels, except for some TSSs in 5’-UTRs. Enhancers are expressed at much lower levels than TSSs.\n\nAnalysing TSS shapes and sequences. A classic analysis of CAGE data is to divide TSSs into Sharp and Broad classes, which show different core promoter regions and different expression patterns across tissues7.\n\nCAGEfightR can calculate several shape statistics that summarizes the shape of a TSS. The Interquartile Range (IQR) can be used to find sharp and broad TSSs. As lowly expressed TSSs cannot show much variation in shape due to their low width and number of tags, we here focused on highly expressed TSSs (average TPM >= 10):\n\n\n\nWe can then plot the bimodal distribution of IQRs. We use a zoom-in panel to highlight the distinction between the two classes:\n\n\n\nWe see most TSSs are either below or above 10 bp IQR (dashed line), so we use this cutoff to classify TSSs into Sharp and Broad:\n\n\n\nWe can now investigate the core promoters sequences of the two classes of TSSs. We first need to extract the sequences for each TSS: We define this as the TSS peak -40/+10 bp and extract them from using the BSgenome.Mmusculus.UCSC.mm10 genome package:\n\n\n\nThis returns a DNAStringSet-object which we can plot as a sequence logo32 via the ggseqlogo package33:\n\n\n\nAs expected, we observe that Sharp TSSs tend to have a stronger TATA-box upstream of the TSS compared to Broad TSSs.\n\nFinding candidates for interacting TSSs and enhancers. In addition to simply identifying enhancers, it is also interesting to try infer what genes they might be regulating. CAGE data can itself not provide direct evidence that an enhancer is physically interacting with a TSSs, which would requires specialized chromatin confirmation capture assays such as HiC, 4C, 5C, etc. However, previous studies have shown that TSSs and enhancers that are close to each other and have highly correlated expression are more likely to be interacting. We can therefore use distance and correlation of expression between TSSs and enhancers to identify TSSs-enhancer links as candidates for physical interactions13.\n\nTo do this with CAGEfightR, we first need to indicate the two types of clusters as a factor with two levels:\n\n\n\nWe can then calculate all pairwise correlations between TSSs and enhancer within a distance of 50 bp. Here we use the non-parametric Kendall’s tau as a measure of correlation, but other functions for calculating correlation can be supplied (e.g. one could calculate Pearson’s r on log-transformed TPM values to only capture linear relationships):\n\n\n\nThe output is a GInteractions-object from the InteractionSet package23: For each TSS-enhancer both the distance and orientation (upstream/downstream relative to TSS) is calculated in addition to the correlation estimate and p-value. For now, we are only interested in positive correlations, so we subset and sort the links:\n\n\n\n\n\nWe can then visualize the correlation patterns across a genomic region, here using the most correlated TSS- enhancer link:\n\n\n\nThe top track shows the strength of correlations between 3 TSSs around the Atp1b1 gene. The highest correlation is seen between the upstream TSS and the most distal enhancer.\n\nFinding stretches of enhancers. Several studies have found that groups or stretches of closely spaced enhancers tend to show different chromatin characteristics and functions compared to singleton enhancers. Such groups of are often referred to as “super enhancers” or “stretch enhancers”34.\n\nCAGEfightR can detect such enhancer stretches based on CAGE data. CAGEfightR groups nearby enhancers into groups and calculates the average pairwise correlation between them, shown below (again using Kendall’s tau):\n\n\n\nSimilarly to TSSs and enhancers, we can also annotate stretches based on their relation with known transcripts:\n\n\n\nThe returned GRanges contains the the location, number of enhancers and average correlation for each stretch. Stretches are found in a variety of context, some being intergenic and other spanning various parts of genes. Let us plot one of the top intergenic stretches:\n\n\n\nThis stretch is composed of at least 5 enhancers, each of which shows bidirectional transcription.\n\nNormalization of expression and EDA. Before performing statistical tests for various measures of Differential Expression (DE), it is important to first conduct a thorough Exploratory Data Analysis (EDA) to identify what factor we need to include in the final model.\n\nHere we will use DESeq220 for normalization and EDA since it offers easy to use functions for performing basic analyses. Other popular tools such as edgeR21 and limma25 offer similar functionality, as well as more specialized packages for EDA such as EDASeq.\n\nDESeq2 offers sophisticated normalization and transformation of count data in the form of the variance stabilized transformation: this adds a dynamic pseudo-count to normalized expression values before log transforming to dampen the inherent mean-variance relationship of count data. This is particularly useful for CAGE data, as CAGE can detect even very lowly expressed TSSs and enhancers.\n\nFirst, we fit a “blind” version of the variance-stabilizing transformation, since we do not yet know what design is appropriate for this particular study:\n\n\n\nA very useful first representation is a Principal Components Analysis (PCA) plot summarizing variance across the entire experiment:\n\n\n\nWe observe that PC2 separates the samples according to the experimental group (control vs nano). However, PC1 also separates samples into two groups. This is suggestive of an unwanted yet systematic effect on expression, often referred as a batch effect. We do not want to mistake this unwanted variation for biological variation when we test for differential expression. To prevent this, we can include the batch information as a factor in the final model. Let first define the batch variable:\n\n\n\nAn alternative to manually defining the batch variable, tools such as sva and RUVSeq can be used to estimate unknown batch effects from the data.\n\nCluster-level differential expression. Following our short EDA above, we are ready to specify the final design for the experiment: We want to take into account both the Class and Batch of samples:\n\n\n\nWe can now extract estimated effects (log fold changes) and statistical significance (p-values) for the Nanovs-Ctrl comparison, implicitly correcting for the batch effect:\n\n\n\nIt always a good idea to inspect a few diagnostics plot to make sure the DESeq2 analysis was successful. One such example is an MA-plot (another useful plot is p-value histogram):\n\n\n\nWe can see that we overall find more differentially expressed TSSs compared to enhancers, which is expected since they are also more highly expressed. Many enhancers are filtered away for the final DESeq2 analysis (The “Independent Filtering” Step), as their expression level is too low to detect any DE: This increases power for detecting DE at higher expression levels.\n\nWe can tabulate the total number of DE TSSs and enhancers:\n\n\n\nCorrecting expression estimates for batch effects. In addition to looking at estimates and significance for each cluster, we might also want to look at individual expression values for some top hits. However, we then need to also correct the expression estimates themselves for batch effects, just like we did for log fold changes and p-values (using the same model of course).\n\nHere we use ComBat35 from the sva package which is suitable for removing simple batch effects from small experiments. For more advanced setups, removeBatchEffect from limma can remove arbitrarily complex batch effects. The RUVSeq package and fsva from sva can be used to remove unknown batch effects.\n\nWe again use the variance-stabilizing transformation to prepare the data for ComBat (this makes count data resemble expression estimates obtained from microarrays, as ComBat was originally developed for microarrays).\n\n\n\nTo run ComBat we need two additional pieces of information: i) A design matrix describing the biological or wanted effects and ii) the known but unwanted batch effect. We first specify the design matrix, and then run ComBat:\n\n\n\nLet us redo the PCA-plot, to see the global effect of the batch effect correction:\n\n\n\nNow Nano and Ctrl are separated along the first principal component (compared to the second principle component before correction).\n\nThen we extract the top 10 DE enhancers using the following tidyverse code:\n\n\n\nFinally, we can plot the batch-corrected expression profiles of each individual enhancer:\n\n\n\nEnrichment of DNA-binding motifs. A typical question following identification of differentially expressed TSSs and enhancers, is what TFs might be involved in their regulation. To shed light on this question we can annotate TSSs and enhancers with DNA-binding motifs from the JASPAR database27.\n\nFirst we extract the sequences around TSSs and enhancers. Here we simply define it as +/- 500 bp around TSS peak or enhancer midpoint:\n\n\n\nSecondly, we use the TFBSTools36 package to obtain motifs as Position Frequency Matrices (PFMs) from the JASPAR2016 database:\n\n\n\nThirdly, we use the motifmatchr package37 to find hits in the sequences:\n\n\n\nFinally we can do a simple Fisher’s Exact test to see if a motif co-occurs more with DE TSSs and enhancer than we would expect be chance. Here we will look at the FOS::JUN motif (MA0099.2):\n\n\n\nA significant odds ratio above 1 indicate that FOS::JUN is a candidate transcription factor (or, more technically correct, a candidate transcription factor dimer) in regulation of the nanotube response. This is not surprising given that FOS::JUN is part of the TNF-alpha inflammatory pathway (see more below).\n\nOf course, this is a just a very quick and simple analysis of motif enrichment. One could easily have used different regions around TSSs and enhancers and/or split the enrichment analysis between TSSs and enhancers. Other Bioconductor packages like PWMEnrich, rGADEM and motifcounter implements more advanced statistical methods for calculating enrichment of known motifs. rGADEM, BCRANK and motifRG can also be used to calculate enrichment of novel motifs, sometimes referred to as motif discovery.\n\nGene-level differential expression. While CAGE data is naturally analyzed at the level of clusters (TSSs and enhancers) it is in many cases interesting to also look at gene-level expression estimates. A prime example of this is looking at enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms28,29,30 which are only defined at gene-level. CAGEfightR includes functions for annotating clusters with gene models and summarizing expression to gene-level.\n\nWe can annotate clusters with gene IDs in the same manner as Transcript IDs:\n\n\n\nAnd then use CAGEfightR to sum counts of TSSs within genes:\n\n\n\nThe result is RangedSummarizedExperiment where the ranges are a GRangesList holding the TSSs that were summed within each gene:\n\n\n\nThe gene IDs in this case is Entrez ID (which is widely used by Bioconductor packages). We can translate these systematic IDs into more human-readable symbols using the org.Mm.eg.db annotation package:\n\n\n\nHaving obtained a gene-level count matrix we can now perform gene-level DE analysis. Here we use limma-voom, since limma makes it easy to perform a subsequent enrichment analysis. Other tools such as DESeq2 (above) or edgeR (see below) could also have been used.\n\nNote: limma is a powerful tool for DE analysis of count-based data. However, since it depends on log transforming counts, it is not always suitable for analyzing datasets where features have very low counts. This is usually not a problem for gene-level analysis, but can be a problem for enhancers, which are generally very lowly expressed.\n\nSimilarly to the DESeq2 analysis, we first build the necessary object and then normalize the expression values:\n\n\n\nThen we apply the voom-transformation to model the mean-variance trend, for which we also need to specify the design matrix (in this case the design must contain both wanted and unwanted effects!). The same design matrix is then used for fitting the gene-wise models:\n\n\n\nWe can the both report the overall summary of differential gene expression, and look at the first few top hits:\n\n\n\n\n\nEnrichment of GO- and KEGG-terms. In addition to looking at individual top genes, we can look at how the differentially expressed genes relate to known databases of gene function to gain insight in what biological processes might be affected in the experiment.\n\nlimma makes it easy to perform such an enrichment analysis following a DE analysis. As we have gene indexed by Entrez IDs, we can directly use goana to find enriched GO-terms: goana uses a biased urn-model to estimate enrichment of GO-terms, while taking into account the expression levels of DE genes:\n\n\n\nAnd similarly for KEGG terms:\n\n\n\nBoth analyses indicate that genes related to the inflammatory response and defense response are upregulated following nanotube exposure. This supports the hypothesis that nanotube induces a response similar to asbestos.\n\nKEGG-terms represents well defined pathways. We can use the pathview package38 to investigate in more detail the genes in a given enriched pathway. For example, we can look at regulation of gene in the TNF- signalling pathway:\n\n\n\nDifferential TSS Usage. In the above two analyses we looked at whether an individual TSSs or an individual gene was changing expression between experimental groups. However, we might also want to look at whether a gene show differential TSS usage: whether a gene uses different TSSs under different conditions. This problem is similar to differential splicing in RNA-Seq, but looking at TSSs rather than isoforms26. Here we will use the edgeR diffSpliceDGE method to find differential TSS usage, although many other packages could have been used, for example diffSplice from limma, DEXSeq, DRIMSeq, etc..\n\nIntuitively, diffSpliceDGE tests whether a given TSSs show the same change as other TSSs in the same gene, indicating that TSSs are differentially regulated across the gene. This does however not take into account the relative composition of a given TSSs, e.g. whether a TSS increases from 1%–2% of gene output or 25%–50%. A useful preprocessing step is therefore to filter out TSSs making only a small contribution to total gene expression before analyses.\n\nWe use CAGEfightR to remove TSSs that are not expressed as more than 10% of total gene expression in more than 5 samples (We first remove TSSs not assigned to genes):\n\n\n\nWe can only do differential TSS usage analysis of genes with multiple TSSs. A useful first visualization is therefore to see how many genes use more than one TSS:\n\n\n\nWhile most genes utilize only a single TSSs, many genes use two or more TSSs.\n\nAgain, we build the necessary R-objects for running edgeR:\n\n\n\nThen we normalize and fit models using the Quasi-likelihood approach, including the diffSpliceDGE step:\n\n\n\nNow we can look at differential TSS usage at two-levels: Whether an individual TSS shows differential TSS usage (TSS-level) or whether a gene show differential TSS usage in any way (gene-level). First we can look at individual TSSs (TSS-level differential TSS usage):\n\n\n\nThe interpretation of log fold changes here is slightly different from before: These log fold changes are relative to the overall log fold change for all TSSs in that gene.\n\nThen we can look at results for each gene (Gene-level differential TSS usage):\n\n\n\nWe see that the two lists agree, which is not surprising given that the gene-level results are obtained by aggregating TSS-level p-values across genes.\n\nWe can look at closer at the TSS usage in on of the top hits: We can visualize the batch-corrected expression (See above) of each TSS in the Fblim1 gene via a heatmap:\n\n\n\nFblim1 has 3 TSSs, with 2 of them being used in the Ctrl samples, while the Nano samples also uses the chr4:141154044-141154185;- TSS, as also seen in the TSS-level table above. While a heatmap is useful for seeing expression changes, a genome browser view is better to inspect the genomic context of each TSSs:\n\n\n\nThe Fblim1 gene uses two annotated TSSs, but the Nano samples also uses a novel intronic TSS.\n\n\nDiscussion\n\nThis workflow is intended as providing an outline of the basic building blocks of CAGE data analysis, going from clustering, to spatial analyses to differential expression. More advanced analyses can be strung together from these basic elements: Finding enhancers linked to DE TSSs, enhancer stretches composed of DE enhancer, comparing DNA binding motif enrichments between DE enhancers and TSSs, etc.\n\nOne aspect not covered in this workflow is the utility of CAGE data (and 5’-end data in general) in providing accurate TSSs for studying other types of data. For example, having accurate TSSs is highly beneficial in chromatin research, since the location and nucleosome and TSSs are closely related13,39,40. CAGE can be combined with chromatin confirmation assays such as HiC to find new enhancers that are both co-expressed and physically interacting with TSSs. Many genome-wide association studies are finding that disease-related genetic variants are found in intergenic regions, that are often poorly annotated. The accurate enhancer locations provided by CAGE can greatly aid interpretation of such variants41. The adherence of CAGEfightR to standard Bioconductor classes facilitates these inter-assay analyses by making it easy to mix-and-match multiple packages developed for different experimental assays.\n\n\nSoftware and data availability\n\nThe following software versions were used in this article:\n\nR version: R version 3.6.0 (2019-04-26)\n\nBioconductor version: 3.9\n\nCAGEfightR version: 1.4.0\n\nCAGEWorkflow: https://doi.org/10.18129/B9.bioc.CAGEWorkflow42\n\nLicense: GPL-3\n\nMouse nanotube CAGE data: https://doi.org/10.18129/B9.bioc.nanotubes24\n\nLicense: GPL-3", "appendix": "Grant information\n\nWork in the Sandelin Lab was supported by the Novo Nordisk Foundation, Lundbeck foundation, Danish Innovation Fund, Danish Cancer Society and Independent Research Fund Denmark.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe acknowledge all members of the Sandelin Lab and Andersson Lab for advice, discussion and input on all aspects related to CAGE data analysis.\n\n\nReferences\n\nSmale ST, Kadonaga JT: The RNA polymerase II core promoter. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagih O: ggseqlogo: a versatile R package for drawing sequence logos. Bioinformatics. 2017; 33(22): 3645–3647. PubMed Abstract \| Publisher Full Text\n\nPott S, Lieb JD: What are super-enhancers? Nat Genet. 2015; 47(1): 8–12. PubMed Abstract \| Publisher Full Text\n\nJohnson WE, Li C, Rabinovic A: Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics. 2007; 8(1): 118–27. PubMed Abstract \| Publisher Full Text\n\nTan G, Lenhard B: TFBSTools: an R/bioconductor package for transcription factor binding site analysis. Bioinformatics. 2016; 32(10): 1555–1556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchep A: motifmatchr: Fast Motif Matching in R. 2018. Publisher Full Text\n\nLuo W, Brouwer C: Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics. 2013; 29(14): 1830–1831. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuttke SHC, Lacadie SA, Ibrahim MM, et al.: Human promoters are intrinsically directional. Mol Cell. 2015; 57(4): 674–684. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThodberg M, Thieffry A, Bornholdt J, et al.: Comprehensive profiling of the fission yeast transcription start site activity during stress and media response. Nucleic Acids Res. 2019; 47(4): 1671–1691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoyd M, Thodberg M, Vitezic M, et al.: Characterization of the enhancer and promoter landscape of inflammatory bowel disease from human colon biopsies. Nat Commun. 2018; 9(1): 1661. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThodberg M: CAGEWorkflow: A step-by-step guide to analyzing CAGE data using R/Bioconductor. R package version 1.0.0. 2019. Publisher Full Text" }	[ { "id": "50074", "date": "24 Jun 2019", "name": "Aaron T. L. Lun", "expertise": [ "Reviewer Expertise Bioinformatics", "computational 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\nThodberg and Sandelin describe a comprehensive workflow for the basic analysis of CAGE data. The workflow is simple, well-presented and the code mostly runs without problems. I only have a few minor comments to improve its usefulness for the general reader:\nThere should be a general overview on how the CTSS BigWig files are generated from raw FASTQ files.\n\nConsider using ExperimentHub for the nanotubes package. This provides greater control over which files are downloaded, rather than forcing the user to obtain all files at installation. This is especially useful if you have multiple data sets - see, for example, the chipseqDBData package.1\n\nExplain what a BigWigFileList is, and why it needs to be used instead of having a simple character vector.\n\nConsider not having string'ified intervals as the row names in the output of quickTSSs(). In some situations, generation of the strings can use more RAM than the actual data itself. It definitely slows down any attempt to 'show' the output. I would suggest that this be deferred as late as possible, e.g., until someone needs the strings as row names of a data frame to save to file.\n\nMore details are required on how quantification is performed. For example, I assume counts are summed directly from single strands for TSSs. For enhancers, are counts summed from both strands?\n\n\"As enhancers are the most complicated type, we keep only enhancers if a TSS and enhancer overlaps:\" The complexity of the enhancers doesn't really provide a motivation for only keeping enhancers in cases of overlaps. The better reason is that all enhancers would be detected as two TSSs if the strandedness was ignored; if they do overlap, it would be more appropriate to interpret them as a single enhancer rather than as two distinct TSS events.\n\nThe Interquartile Range (IQR)... of what? I assume that the range refers to the length of the interval that contains 10 to 90% of a TSS's counts. Incidentally, the IQR is no longer an IQR if it's redefined from 10-90%.\n\nThe single quotes in the highTSSs call are malformed, which prevents copy-pasting.\n\nOne could consider using a 2-component mixture model to classify elements into sharp/broad in a more automated manner.\n\nI presume that the pairwise correlations for the TSS-enhancer interactions are computed across samples for each TSS/enhancer pair. If so, is this done after blocking on the design? Otherwise it is possible to obtain strong positive correlations simply because a TSS and the enhancer happen to respond in the same direction to a given treatment. If there is a genuine physical interaction, it should manifest as correlations within each treatment condition, where stochastic differences in RNA polymerase activity affect both the TSS and its interacting enhancer.\n\nThere is no correction for multiple testing in the p-values from the links. While I recognise that this is a limitation of the small sample size, it should still be pointed out as a caveat of the analysis.\n\nAll normalization steps in the DE analyses assume that most of the input features are not differentially expressed between conditions. This is usually not a concern, but if aggressive feature selection is performed, it may become a problem. For example, if one were to perform the DE analysis on superenhancers only, it would give incorrect results in situations where the superenhancer activity increases globally upon treatment.\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": "50076", "date": "05 Jul 2019", "name": "Kenta Nakai", "expertise": [ "Reviewer Expertise bioinformatics", "genome sequence analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors present a cookbook for analyzing CAGE data mainly through their own Bioconductor package, CAGEfightR, applied to sample data, which were analyzed and published by their group previously. By following the given source codes, readers will be able to learn how they can obtain various kinds of information rather easily. Thus, I feel that this publication will be useful for those who want to learn how to analyze CAGE data quickly. On the other hand, it will not tell us how to solve our deeper problems in research. I know that this is out of the scope of this tutorial but I can’t help but feeling, for example, how the given procedure for reducing batch effects can be justified (see below). Thus, here are some of my suggestions for its further improvement:\nIn our realistic situations, the EDA approach is quite important. In this sense, I appreciate their demonstration on how to remove batch effects from the expression data between various samples, using ComBat (Figs. 9 and 11). However, most researchers will not be satisfied with just seeing that the PC1 has become to separate positive and negative groups; it is natural that they would like to confirm if the correction was enough or not; they would also wish to see what the new PC2 as well as the old PC1 represent. Therefore, I recommend the authors to extend Table 2 for characterizing each sample from various features (e.g., experimental conditions and data size) and to use such features for the interpretation. More discussion and/or additional attempts to clarify the most probable main reason for the initial batch effects would be desirable. Similarly, since the first author does not seem to have been a member of the previous analysis, it is interesting to see the consistency between the two studies. For example, the observation that inflammation-related genes were activated seems to be the same in both analyses. Then, are the genes with differential TSSs likely to explain the phenomenon? How much are the detected enhancers contributing to the differential expression? Do these enhancers (or newly activated TSSs) share any over-represented motifs? From the same reason, I recommend the authors to avoid using (ugly) chromosomal coordinates to represent genes/promoters/enhancers, wherever possible. It would be great if the authors can show that they could perform deeper analyses this time. For the convenience of wider readers, it might be useful to show the way how to obtain BigWig files from rawer data (BAM or fastq, if possible). Similarly, a summary table of used tools (except CAGEfightR), containing their input file information as well as their main purposes might be useful. Also, it might be useful if there is a summary on what CAGEfightR can do/cannot do. For example, is it possible to combine different sources of CAGE data with this workflow? One of my students tried to follow the workflow. At first, she failed to install some of the packages. It was because the version of R she used was R3.5. Thus, this point should be clearly noted. In addition, she reports that to run the code “Fit and shrink DE model”, the installation of “statmod” and “BiasedUrn” was necessary. Perhaps, some additional information on how to setup initial environment would be useful for beginners. As a cookbook, it is desirable that users can find their necessary items more easily (via a table of contents with clearer headers, perhaps?). There seems to be some confusion on the versions used: As for the mouse genome sequence, (1) both mm9 and mm10 are used. (2) Why didn’t they use the latest version of JASPAR (Jaspar2018)? The manuscript seems to contain many typos. Here are some that we found (there are likely to be more): (1)This is workflow is a case study on (2) CAGE dusters (3) can be speed up (4) novel TSSs are expresse d (5) to try infer (6 )this is a just a very quick (7) The returned GRanges contains the the location (8) going from clustering, to spatial analyses to differential expression.\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? 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": "50326", "date": "10 Jul 2019", "name": "Nevena Cvetesic", "expertise": [ "Reviewer Expertise Computational and experimental genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThodberg and Sandelin present CAGEfightR, a new Bioconductor package suited for analysis of 5'end oriented datasets, derived from CAGE-seq and similar techniques. Although similar Bioconductor packages exist (icetea, CAGEr, TSRchitect), the greatest strength of CAGEfightR is unique in its ability to call or predict putative enhancers based on the bidirectional transcription initiation signature, thus filling the gap in the current R-based toolkit for CAGE-like data analysis. In addition, using CAGEfightR, hypotheses of enhancer-promoter interactions based on co-expression levels can be easily set and visualised.\nThe package is well-documented, and the step-by-step protocol well written and easy to follow. I only have a few minor comments which could benefit the general reader:\nThe introduction and abstract could state more clearly that CAGE allows TSS mapping of only RNA polymerase II transcripts. Though this is implied through usage of cap-trapping, I would keep in mind that this workflow might be used by general readers not so familiar with TSS-mapping techniques. Considering the advent of technologies that capture RNA polI-RNApolIII transcripts, expected to be much noisier, it would be better to make it as clear as possible. If the authors believe CAGEfightR could be of use on noisier data, from experimental methods based on negative selection (such as TSS-seq), it would be worth testing this and including a few sentences, as this would promote CAGEfightR usage on any TSS mapping technique.\n\nI support comparison of existing packages in the form of Table 1; however, I would expand this comparison to include unique features that perhaps CAGEfightR does not have - e.g. icetea and TSRchitect support paired-end data, CAGEr has TSS-shifting discovery function and implemented G-correction function to remove mismatching G’s from 5’ends of reads.\n\nCommon problem with CAGE and CAGE-like data which is obtained through reverse transcription, is the addition of a G, or so called G-bias upstream of the true transcription start site. It would be beneficial for general readers as this is a step-by-step protocol to discuss how to generate BigWig files from fastq files, and how to address the G-bias problem/i.e. remove the mismatching G’s at the 5’end of the reads.\n\nThe authors prefer to use the term TSS regions or TSSs in place of tag clusters as aggregated CTSSs, and even TSSs as a broader term for CTSS while CTSS is just a CAGE-supported TSS. This becomes very confusing for a general reader, especially page 36: “Now we look at differential TSS usage at two-levels: Whether an individual TSS shows differential TSS usage (TSS-level) or whether a gene show differential TSS usage in any way (gene-level). First we can look at individual TSSs (TSS-level differential TSS usage).” I would suggest usage of CTSS for individual CAGE-supported TSSs, tag cluster - for an aggregate of individual CTSSs based on distance based clustering or whatever methodology, and tag cluster can be interchangeably used with promoter where needed.\n\nInterquartile range should probably be interquantile range as it spans middle 10-90th percentile of the signal. It would also be beneficial to explain why is it used instead of all signal (more robust measure that excludes outliers and is less sensitive to sequencing depth etc).\n\nI am a bit surprised the authors use such harsh filtering step before plotting distributions of IQR (Figure 5, TPM >=10), I would assume that the problem is in tag clusters which seem sharp - single bp, and therefore it would perhaps be beneficial to add a more stringent filtering step only to single bp tag clusters to be retained only if highly expressed (>= 5 TPM), while a lower threshold can be applied on broad tag clusters as multiple CTSS within a tag cluster give more certainty that it is not just noise.\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/8-886
https://f1000research.com/articles/7-1823/v1	20 Nov 18	{ "type": "Research Article", "title": "Distribution of Toxoplasma gondii IgM and IgG antibody seropositivity among age groups and gestational periods in pregnant women", "authors": [ "Shahida Sadiqui", "Syed Rafiq Hussain Shah", "Babiker Saad Almugadam", "Qismat Shakeela", "Shehzad Ahmad", "Shahida Sadiqui", "Babiker Saad Almugadam", "Qismat Shakeela", "Shehzad Ahmad" ], "abstract": "Background: Toxoplasmosis is a globally distributed parasitic disease. The present study aimed to estimate the prevalence and geographic distribution of toxoplasmosis, as well assess the risk of animal contact in disease development and determine the percentage of toxoplasmois-associated IgM and IgG seropositivity among different age groups. In addition, it aimed to estimate the proportion of toxoplasma IgM seropositivity among pregnancy trimesters. Methods: A total of 500 pregnant women were included in this study. From each participant, a 5-ml venous blood sample was collected and centrifuged to obtain serum that was tested for Toxoplasma gondii IgM and IgG antibodies using immunochromatographic testing and ELISA. Results: The overall seroprevalence of toxoplasmosis was 24.8%, with rates of acute infection of 8%. Among positive cases in every trimester, 54.34% of first trimester positive cases had a serologic marker of acute toxoplasmosis. Out of the 40 pregnant women with previous history of cow/buffalo contact and toxoplasmosis, 40% were seropositive for toxoplasma IgM; and out of 30 women with prior history of dog contact, 16.66% had serological marker of acute toxoplasmosis. Conclusions: In this study, there is a high prevalence of toxoplasmosis and contact with domestic animals is a risk factor for this illness. Therefore, it is necessary to test every pregnant women for toxoplasmosis and distinguish the type of infection, as well as the conduction of public health education programs to generate the awareness.", "keywords": [ "Toxoplasma gondii", "Toxoplasmosis", "Seroprevalence", "IgG", "IgM", "Pregnant women" ], "content": "Introduction\n\nToxoplasmosis is a widely distributed zoonotic illness caused by Toxoplasma gondii, an obligate intracellular parasite1,2. Globally, the distribution of this disease is extremely variable even inside the countries3,4. In all host species, including humans, Toxoplasmosis is generally acquired either vertically from mother to fetus (congenital infection), or through ingestion of oocysts in contaminated food or water5. Rarely, T. gondii can transmit through organ transplantation and the transfusion of infected blood6,7. Following ingestion, the intestinal epithelium is the primary portal of entrance for T. gondii; next, it spreads to other tissues, where it can cause more severe pathogenesis8,9. If toxoplasmosis is acquired during pregnancy, severe infection may develop, especially in immunocompromised individuals, such as those with defects in T-cell-mediated immunity10. In patients with AIDS, toxoplasmosis may leads to severe, life-threatening disease11. For example, cerebral focal lesions are caused by cerebral toxoplasmosis (CT) in HIV-infected patients12.\n\nThe signs and symptoms of this illness are markedly divergent and range from asymptomatic to serious infection13. This variation depends on several factors includes inoculums size, virulence of the strain of toxoplasma, the individual’s genetic background and the status of the immune system of the infected individual14. In addition, since the organism has an affinity for muscular and neural tissues as well as the other visceral organs, many hosts harboring latent tissue cysts following toxoplasmosis15.\n\nFetuses may acquire toxopasmosis during delivery or through the placenta during pregnancy16. Early infection of the fetus may cause severe damage, or either pre- or post-natal death17. The clinical manifestation of congenital toxoplasmosis generally depends on the gestational stage, and can includes seizures, mental retardation, severe neurological defects, chorioretinitis, epilepsy and blindness10,16,18.\n\nApproximately 90% of pregnant women infected with T. gondii are asymptomatic, and recover spontaneously19,20. Only a small percentage of pregnant women show the clinical symptoms of disease19,21. In pregnant women, the clinical signs are no more severe than in non-pregnant women, and typically an influenza-like illness is seen after an incubation period of 5 to 18 days19,22,23. Early diagnosis and treatment of mothers during pregnancy prevents fetal infection and minimizes the probability of complications24,25.\n\nLaboratory diagnosis of toxoplasmosis is usually performed by serological detection of T. gondii-specific IgG and IgM antibodies26. Worldwide, the screening of T. gondii infection in pregnant women is preferably performed during the first trimester and subsequently every month or trimester in seronegative women, as applied in many countries27.\n\nOur study was undertaken to determine the prevalence and geographic distribution of toxoplasmosis. In addition, it sought to evaluate the role of animal contact in disease development among pregnant women through the serological detection of toxoplasma IgM and IgG antibodies, as well as to estimate the seropositivity of these antibodies among different age groups. It also attempted to identify the percentage of toxoplasma IgM seropositivity (indicative of acute infection) among different pregnancy trimesters.\n\n\nMethods\n\nThis was a descriptive cross-sectional hospital-based study carried out in the District Head quarter Hospital (Mansehra, Hazara, Pakistan) and Ayub Medical Complex Hospital (Abbottabad, Khyber Pakhtunkhwa, Pakistan) over a period of 4 months (April to July 2015).\n\nOur study included pregnant women of different trimesters, ages and ethnic groups who visited our study areas hospitals; the only eligibility criteria were pregnancy and visiting the hospitals in our study area. Patients were recruited by the researchers face-to-face. During this study duration, a total of 500 pregnant women (convenience sample) fulfilled the inclusion criteria. Out of the total of participants, 204 were recruited from Abbottabad and 296 from Mansehra district.\n\nA total of 5 ml venous blood was collected from each participant using a sterile syringe and transferred to a blood container without anticoagulant, allowed to clot at room temperature for 15 minutes, then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was transferred into a 1.5-ml microcentrifuge tube and stored at −80°C for further analysis. In this study, every sample was screened and confirmed for toxoplasmosis through the serological tests.\n\nAll sera samples were screened for T. gondii IgG and IgM antibodies using Rapid Diagnostic immunochromatographic test (Tox IgG/IgM Rapid Test Dip strip, CTK BIOTECH, San Diego, USA) according to manufacture instructions. In order to avoid false-positive results due to the incomplete specificity of the screening test, every positive sample was further subject to confirmation step by ELISA. Each positive individual also answered a questionnaire concerning their age, trimester and whether they had been in recent contact with animals (Supplementary File 1).\n\nFollowing screening, the positive samples (n=150) were further confirmed to toxoplasmosis using IgM and IgG ELISA kit (Monobind, San Diego, USA) according to the manufacturer’s protocol. The samples positive for T. gondii IgG titers indicated chronic infection and those with high IgM titers indicates recent or acute infection. All ELISA tests were performed in triplicate.\n\nOur study was approved by Hazara University. Further approval was provided by the Ayub Medical Complex Hospital. From every participant, written informed consent was obtained for conduction of the study. In addition, all the performed steps in this study were completely in accordance with Helsinki Declaration and the rules defined by the World Medical Association, including samples collection and processing.\n\nThe obtained results were analyzed by Graph Pad Prism 5 (Graph Pad Software, La Jolla, CA, USA). A χ2 test was involved to test the statistical differences in seropositivity and negativity of anti-toxoplasma antibodies among the participants of different study areas and gestational periods or those had/had no prior history of animal contact, at 95% level of significance. Moreevore, ANOVA was tested the statistical difference of these antibodies among the participants of every age group. Difference was considered statistically significant when P <0.05.\n\n\nResults\n\nOut of 500 women, using ELISA the overall seroprevalence of toxoplasmosis was 24.8% (124/500). Statistically significant differences were observed between the seroprevalence of disease in Abbottabad and Mansehra district (Figure 1). In addition, the prevalence of toxoplasma antibodies among pregnant women revealed out of the total of 500 participants, only 8% had a serological marker of acute toxoplasmosis (Figure 2).\n\nOut of the total of participants in every district, 38.7% (79/204) had the serologic marker of toxoplasmosis in Abbottabad district and 15% (45/296) in Mansehra. *P = 0.0002.\n\nOut of 500 pregnant women, 8% (40/500) were positive to IgM, 10.8% (54/500) to IgG, and 6% (30/500) to both antibodies. P = 0.567.\n\nAmong the positive cases (n=124), the seropositivity of toxoplasma antibodies was shown to be statistically significant different among different age groups (Table 1). There was also a statistically significant difference in the seropositivity of toxoplasma IgM (indicating acute infection) between different gestational trimesters, the highest level of IgM seropositivity was observed in first trimester (54.34%) (Figure 3).\n\nAmong the total of positive cases in every trimester, the seropositivity of IgM revealed statistically significant difference. Out of 46, 51, and 27 toxoplamosis infected cases in first, second and third trimesters, respectively, 54.34% (25/46) were seropositive to IgM (acute infection) in first trimester, 21.56% (11/51) seropositive to IgM in second trimester, and 14.81% (4/27) seropositive to IgM in third trimester. **P = 0.0001.\n\nOur study findings revealed the previous animal contact is associated with toxoplasmosis (Table 2). In addition, the occurrence of Toxoplasmosis is also influenced by the species of animal to a statistically significant level. As we displayed in Table 2, out of 40 women with previous history of cow/buffalo contact, 40% were seropositive for toxoplasma IgM. As well as, out of 30 women with previous history of dog contact, 16.66% had serologic marker of acute toxoplasmosis.\n\n\nDiscussion\n\nToxoplasmosis in pregnancy can predispose the fetus to serious complications28. The fetus can be severely damaged when the infection is acquired during pregnancy29. Therefore, testing the serum of pregnant women for toxoplasma IgG and IgM is important to avoid intrauterine infection and complications. The current study was conducted on 500 blood samples collected from pregnant women in Mansehra and Abbottabad district of Pakistan, and examined for T. gondii IgM (acute infection) and IgG (chronic infection) antibodies. Out of the total of 500 pregnant women, 24.8% (124 women) had serologic marker of toxoplasmosis. Among the 124 positive cases, 54 were seropositive for toxoplasma IgG antibody, 40 cases for Toxo-IgM and 30 cases for both IgM and IgG antibody. In addition, out of 500 participants, 8% had a serologic marker of acute toxoplasmosis. In 2007, Obeed reported the prevalence of IgG (chronic infection) and IgM (acute infection) antibodies were 36% and 26.6%, respectively, which are greater than those seen in our study results30. In addition, the seroprevalence of toxoplasmosis in Saudi Arabia was reported as 21.8%31. In pregnant women from South Korea, a low prevalence rate was observed (0.79%)32, with rates of 20% reported in Finland33 and 24% in Prague34. These findings indicate the prevalence of toxoplasmosis is markedly difference in different countries.\n\nMoreover, our study revealed that the geographic distribution of toxoplasmosis is significantly different among the study areas. Out of the 296 participants analyzed from Mansehra and 204 from Abbottabad, the overall prevalence of toxoplasmosis was 15% and 38.7%, respectively. The higher prevalence in Abbottabad when compared with Mansehra may because Abbottabad is an area where agricultural practices are common, and domestic animals like cats and goats were generally kept in or near the homes. Thus, contact with these animals may be the main risk factor of the disease. In addition, low educational and socioeconomic level may have contributed.\n\nIn our study, a high percentage of IgM seropositivity was reported in the 1st trimester, which indicated a high prevalence of acute toxoplasmosis or recent infection in this trimester compared with the others. Furthermore, as reported in this study, there is a mild difference in the seropositivity of toxoplasma antibodies among age groups, which requires further study to assess whether, is there any significant association exists between toxoplasmosis and age.\n\nUsually T. gondii does not cause clinical illness in the majority of animal species35. Human often acquire this infection from animals by ingestion of improperly cooked or raw animal meat, or via consumption of contaminated food and water with animals waste36. However, there is a need for detailed knowledge about the risk factors of toxoplasmosis. Previously, it was reported that there are some of risk factors are associated with toxoplasmosis, such as owning cats37. Our study found that a considerable percentage of acute toxoplasmosis-infected women had a previous history of close contact with animals; for example, 15% had the history of contact with cats, 16.66% with dogs, 35% with goats and 40% with cows/buffalos. These results indicate that contact with domestic animals carry a risk for this disease. Similar results are also reported by many studies38,39.\n\nIn this study, a high prevalence of toxoplasmosis was revealed. Lack of awareness together with contact with domestic animals are potential risk factors in Mansehra and Abbottabad district of Pakistan. Moreover, in the first and second trimester of pregnancy, the prevalence of acute toxoplasmosis seems to be higher compare with third. Thus it is necessary to test every pregnant women for toxoplasmosis and distinguish the type of infection. In addition, urgent treatment and medicine is essential to decrease the risk of intra-uterine infection and congenital toxoplasmosis. Additionally, there is a need to conduct public health education to create greater awareness about the disease, its transmission, symptoms, and prevention. In addition, screening of T. gondii infection and maternal care should be considered as the main stratagem to reduce the risks of congenital toxoplasmosis.\n\n\nData availability\n\nDataset 1. Raw data associated with this study. The results of immunochromatographic and ELISA screening, and history of animal contact are included. DOI: https://doi.org/10.5256/f1000research.15344.d22433540.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting of this work.\n\n\nAcknowledgements\n\nThe authors acknowledge the study participants and staff of District Head Quarter Hospital and Ayub Medical Complex Hospital.\n\n\nSupplementary material\n\nSupplementary File 1. Questionnaire used in this study.\n\nClick here to access the data.\n\n\nReferences\n\nTenter AM, Heckeroth AR, Weiss LM: Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000; 30(12–13): 1217–1258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDubey JP: Toxoplasmosis of animals and humans. 2nd edition. CRC Press; Boca Raton,Florida, U.S.A, 2010; 1–313. Reference Source\n\nErtug S, Okyay P, Turkmen M, et al.: Seroprevalence and risk factors for toxoplasma infection among pregnant women in Aydin province, Turkey. BMC Public Health. 2005; 5: 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDarabus G, Hotea I, Oprescu I, et al.: Toxoplasmosis seroprevalence in cats and sheep in Western Romania. Revue Med Vet. 2011; 162(6): 316–320. Reference Source\n\nDubey J: Toxoplasma gondii. In S. Baron, ed. Medical Microbiology. Texas: University of Texas Medical Branch. 1996; 486–512. PubMed Abstract\n\nSingh S, Nautiyal BL: Seroprevalence of toxoplasmosis in Kumaon region of India. Indian J Med Res. 1991; 93: 247–49. PubMed Abstract\n\nSabin AB: Toxoplasmosis: recently recognized disease. AdvPediatr. 1942; 1: 1–54.\n\nBarragan A, Sibley LD: Migration of Toxoplasma gondii across biological barriers. Trends Microbiol. 2003; 11(9): 426–30. PubMed Abstract \| Publisher Full Text\n\nSilveira C, Belfort R Jr, Muccioli C, et al.: A follow-up study of Toxoplasma gondii infection in southern Brazil. Am J Ophth. 2001; 131(3): 351–4. PubMed Abstract \| Publisher Full Text\n\nHokelek M: Toxoplasmosis. Medicine cited November, Retrieved march 6, 2011. 2006. Reference Source\n\nNissapatorn V, Lee C, Quek K, et al.: Toxoplasmosis in HIV/AIDS patients: a current situation. Jpn J Infect Dis. 2004; 57(4): 160–165. PubMed Abstract\n\nKristiah K: Studies on the epidemiology of toxoplasmosis in South Africa. Johannesburg: University of Witwatersrand. 2009. Reference Source\n\nDubey JP, Lago EG, Gennari SM, et al.: Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology. Parasitology. 2012; 139(11): 1375–1424. PubMed Abstract \| Publisher Full Text\n\nMontoya JG, liesenfeld O: Toxoplasmosis. Lancet. 2004; 363(9425): 1965–1976. PubMed Abstract \| Publisher Full Text\n\nDubey JP: Detection of Toxoplasma gondii oocysts in drinking water. App Env Micro. 1998; 64(6): 2278–2280. PubMed Abstract \| Free Full Text\n\nWu L: Toxoplasmosis Medicine. Cited April 2007. Retrieved April 3. 2011. Reference Source\n\nMuhie Y, Keskes S: Toxoplasmosis: Emerging and Re-Emerging zoonosis. Afr J of App Micro. 2014; 3: 1–11. Reference Source\n\nRothova A: Occular manifestation of toxoplasmosis. Curr open Opthalmol. 2003; 14(6): 384–8. Reference Source\n\nBoyer K, Holfels E, Roizen N, et al.: Risk factors for toxoplasma gondii infection in mothers of infants with congenital toxoplasmosis: implications for prenatal management and screening. Am J Obstet Gynecol. 2005; 192: 564–71. PubMed Abstract \| Publisher Full Text\n\nKravetz JD, Federman DG: Prevention of toxoplasmosis in pregnancy: knowledge of risk factors. Infect Dis Obstet Gynecol. 2005; 13(3): 161–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarlo DP, Romano A, Schimmenti MG, et al.: Materno-fetal Toxoplasma gondii infection: critical review of available diagnostic methods. Inf Med. 2008; 16(1): 28–32. PubMed Abstract\n\nJones J, Lopez A, Wilson M: Congenital toxoplasmosis. Am Fam Physician. 2003; 67(10): 2131–8. PubMed Abstract\n\nStray-Pedersen B: Toxoplasmosis in pregnancy. Baillieres Clin Obstet Gynaecol 1993; 7(1): 107–37. PubMed Abstract\n\nWallon M, Liou C, Garner P, et al.: Congenital toxoplasmosis: systematic review of evidence of efficacy of treatment in pregnancy. BMJ. 1999; 318(7197): 1511–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeyron F, Wallon M, Liou C, et al.: Treatments for toxoplasmosis in pregnancy. Cochrane Database Syst Rev. 2000; I3(2): CD001684. PubMed Abstract \| Publisher Full Text\n\nWeigel RM, Dubey JP, Dyer D, et al.: Risk factors for infection with Toxoplasma gondii for residents and workers on swine farms in Illinois. Am J Trop Med Hyg. 1999; 60(5): 793–8. PubMed Abstract\n\nAvelino MM, Amaral WN, Rodrigues IM, et al.: Congenital toxoplasmosis and prenatal care state programs. BMC Infect Dis. 2014; 14: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHohlfeld P, Daffos F, Thulliez P: Fetal toxoplasmosis: outcome of pregnancy and infant follow-up after in utero treatment. J Pediatr. 1989; 95(5 Pt 1): 765–9. PubMed Abstract \| Publisher Full Text\n\nDubey JP: Toxoplasmosis - a waterborne zoonosis. Vet Parasitol. 2004; 126(1–2): 57–72. PubMed Abstract \| Publisher Full Text\n\nObeed R: Seroepidem-iolgical study of toxoplasmosis among different groups of population in Najaf of city (Thesis). Kufa University. 2007.\n\nAl-Qurashi AR, Ghandour AM, Obied OE, et al.: Seroepidemiological study of Toxoplasma gondii infection in the human population in the Eastern Region. Saudi Med J. 2001; 22(1): 13–18. PubMed Abstract\n\nSong KJ, Shin JC, Shin HJ, et al.: Seroprevalence of toxoplasmosis in Korean pregnant women. Korean J Parasitol. 2005; 43(2): 69–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLappalainen M, Koskela P, Hedman K, et al.: Incidence of primary toxoplasma infections during pregnancy in southern Finland: a prospective cohort study. Scand J Infect Dis. 1992; 24(1): 97–104. PubMed Abstract \| Publisher Full Text\n\nFlegr J, Hrdá S, Kodym P: Influence of latent 'asymptomatic' toxoplasmosis on body weight of pregnant women. Folia Parasitol (Praha). 2005; 52(3): 199–204. PubMed Abstract \| Publisher Full Text\n\nCunningham AA, Buxton D, Thomson KM: An epidemic of toxoplasmosis in a captive colony of squirrel monkeys (Saimiri sciureus). J Comp Pathol. 1992; 107(2): 207–219. PubMed Abstract \| Publisher Full Text\n\nMontoya JG, liesenfeld O: Toxoplasmosis. Lancet. 2004; 363(9425): 1965–1976. PubMed Abstract \| Publisher Full Text\n\nBaril L, Ancelle T, Goulet V: Risk factors for Toxoplasma infection in pregnancy: a case-control study in France. Scand J Infect Dis. 1999; 31(3): 305–9. PubMed Abstract \| Publisher Full Text\n\nElnahas A, Gerais AS, Elbashir MI, et al.: Toxoplasmosis in pregnant Sudanese women. Saudi Med J. 2003; 24(8): 868–70. PubMed Abstract\n\nMorris A, croxson M: Serological evidence of Toxoplasma gondii infection among pregnant women in Auckland. N Z Med J. 2004; 117(1189): U770. PubMed Abstract\n\nSadiqui S, Shah SRH, Almugadam BS, et al.: Dataset 1 in: Distribution of Toxoplasma gondii IgM and IgG antibody seropositivity among age groups and gestational periods in pregnant women. F1000Research. 2018. https://www.doi.org/10.5256/f1000research.15344.d224335" }	[ { "id": "40932", "date": "10 Dec 2018", "name": "Wafa Babiker", "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 article has studied toxoplasmosis, which is considered as one of the most common causes of abortion, stillbirth, intrauterine death, and congenital abnormalities. In addition, it’s a globally distributed disease.\n\nThe paper is well designed and clearly written; the results were presented accurately and the conclusion is supported by study findings. In my opinion, it’s perfect and scientifically acceptable. However for further improvement of the article, the authors should revise the English grammar.\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": "4497", "date": "01 Apr 2019", "name": "Syed Rafiq Hussain Shah", "role": "Author Response", "response": "Dear Dr. Wafa Babiker, We have addressed your suggestion in Version 2. Thank you." } ] }, { "id": "43385", "date": "12 Feb 2019", "name": "Asghar Fazaeli", "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 manuscript is written in a good structure and presents useful data including the rate of anti-Toxoplasma antibodies (IgG and IgM) in sera of pregnant women referred to hospitals of two districts in Pakistan. The manuscript however requires some corrections and modifications as follows:\nThe authors should consider that “toxoplasmosis” is not necessarily “illness or disease” as such they have mentioned in many parts of the manuscript. Toxoplasmosis is an infection which is mostly latent and does not lead to disease or illness in most of immunocompetent individuals. Also, some other issues in the Introduction are scientifically susceptible, e.g. toxoplasma acquisition in fetuses during delivery (mentioned in the 3rd paragraph of the Introduction); to my knowledge, this issue is not documented in the literature.\n\nThe basis of data arrangement, analysis and discussion about percentage of Toxoplasma gondii antibodies in different age groups (Table 1) is not valid. For this issue, all 500 study cases must be primarily divided into different age groups, then the percentage of seropositivity (IgG, IgM, IgG+IgM) in all initial samples of each group should be calculated and consequently compared and analyzed.\n\nThere is no scientific reason to indicate possible relation between Toxoplasma infection and contact with animals like caw, buffalo, goats, and dogs, except for cats which are a definitive host of Toxoplasma and shed oocysts. The only way of passing Toxoplasma infection from these animals to humans, is ingestion of undercooked meats or rarely through contact of open wounds with meats contaminated with bradyziotes, but not other types of contact with these animals or their wastes. So, this variable (animal contact) is not wise to be included in the manuscript and is recommended to be removed from all parts of the manuscript.\n\nMiss-citation is the case for some Toxoplasma facts in the manuscript. For example, cerebral toxoplasmosis is referred to Kristiah (2009), which is not the right citation; while it was originally reported and discussed by other researchers, i.e. Luft and Remington (19881) and Luft and Remington (19922).\n\nEnglish writing correction is required in some parts of the manuscript text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "4487", "date": "01 Apr 2019", "name": "Syed Rafiq Hussain Shah", "role": "Author Response", "response": "Dear Dr. Asghar Fazaeli,Most of your suggestions have been addressed in Version 2, while we didn't address points 2 and 3 due to the following reasons: Because we aimed to estimate the percentage of toxoplasma antibodies seropositivity among positive cases. Therefore, we calculate them by dividing the frequency of Ab seropositivity/Number of positive cases. Because toxoplasmosis is a zoonotic disease, thus it can transmit to humans from animals as we mentioned in Version 1. In addition, in past research, scholars have found that there is an association of cat contact with disease development, while the association of other kinds of animals with disease is poorly understood. Therefore, we aimed to evaluate the role of different kinds of animals in disease development. Thank you." } ] } ]	1	https://f1000research.com/articles/7-1823
https://f1000research.com/articles/8-875/v1	17 Jun 19	{ "type": "Research Article", "title": "Age, prostate volume, prostate-specific antigen and prostate-specific antigen density as predictor factors in results of transrectal ultrasonography-guided prostate biopsy", "authors": [ "Prima Ciko Ade Putra", "Rainy Umbas", "Agus Rizal Ardy Hariandy Hamid", "Chaidir Arif Mochtar", "Prima Ciko Ade Putra", "Agus Rizal Ardy Hariandy Hamid", "Chaidir Arif Mochtar" ], "abstract": "Background: To identify the predictor factors, such as prostate-specific antigen (PSA), age, prostate volume (PV), and PSA density (PSAD) as indications to perform transrectal ultrasonography (TRUS)-guided prostate biopsy in reducing unnecessary biopsies and improving detection rate. Methods: A total of 1232 samples were obtained from the medical records of patients underwent prostate biopsy from January 2008 to December 2013 in Cipto Mangunkusumo Hospital Jakarta. Pre-biopsy data including age, PSA, prostate volume, and PSAD were obtained. The Mann-Whitney U-test and unpaired t-test were conducted on the quantitative variables; a chi-square test was used for qualitative variables. This study also conducted receiver operating characteristic (ROC) curve analysis to determine the cut-off point and the optimum specificity and sensitivity for each variable. Results: Among 1232 patients, 33.5% had a positive biopsy result. The median age and PSA (68 years and 57.45 ng/ml) in the positive biopsy group was higher than in the negative group (65 years and 11.69 ng/ml), p <0.001. PSAD in patients with PSA 4-10 ng/ml, 10-20 ng/ml, and 20 ng/ml (0.20, 0.35, 2.05) in positive group was higher than negative group (0.14, 0.24, 0.53), p <0.001. Those with a positive biopsy result had a lower median PV (42 ml (range, 13.8-208)) compared to those with negative biopsies (55.4 ml) (p <0.001). In ROC curve, PSAD had the highest sensitivity and specificity (81.4% and 82.0%) with a cut-off point of 0.43 ng/ml/ml (p <0.001). Conclusion: The incidence of PCa increased with higher PSA level, older age and lower PV. Utilization of PSAD 0.17 ng/ml/ml as a cut-off point in patients with PSA level between 4-10 ng/ml is recommended to improve PCa detection in Indonesian men.", "keywords": [ "Age", "prostate volume", "prostate specific antigen", "PSA density", "prostate biopsy", "TRUS" ], "content": "Introduction\n\nProstate cancer (PCa) is an important health issue worldwide because of its increasing incidence; it was reported that there are 1.1 million PCa patients worldwide in 20121. The incidence of PCa among Indonesian men was estimated to be 14.8 per 100,000 population and the mortality rate was reported as high as 9.8 per 100,000 population in 20121.\n\nTransrectal ultrasonography (TRUS) guided biopsy plays an important role for PCa detection and has become a standard diagnostic method for PCa detection. It is recommended for men with elevated prostate specific antigen (PSA) levels or abnormal digital rectal examination (DRE). In Indonesia, 66.5% of PCa was diagnosed with TRUS-guided biopsy during 1994–2005 and 66.7% of them were found in advanced stage during this period2,3. An appropriate and early detection are needed to detect PCa in its early stages for better treatment results, survival rates, and quality of life4.\n\nThe detection rate of prostate biopsy in several Asian countries varied between 14.6% and 26.5% in patients with PSA levels ranging from 2–20 ng/ml5. This widely used procedure for PCa remains controversial as it may lead to over-diagnosis (23%–42%) and over-treatment5. Both are related to unnecessary risk of urinary, sexual, and bowel dysfunction, which can affect patients’ quality of life4,6. Although TRUS-guided prostate biopsy is a safe procedure, there are still complications causing significant morbidity. The most common complications are mild hematuria (62%), rectal bleeding (2%), urinary tract infections (1.7%–11.3%), acute urinary retention (0.3%–2.6%), hematospermia (45.3%–50.4%), vasovagal response (2.8%), and hospitalization due to infection (1%–4.1%)7,8). Systematic screening for prostate cancer by determining other factors besides PSA may assist prevention of this circumstance5.\n\nThe aim of this study was to identify the predictor factors such as PSA, age, prostate volume (PV), and PSA density (PSAD) as indications to perform TRUS guided prostate biopsy. Those variables are thought to be important predictors to reduce unnecessary biopsies and improve detection rate.\n\n\nMethods\n\nThis retrospective study was approved by FKUI Research Ethical Committee before data collection began. We collected data pre-biopsy data, including age, PSA, prostate volume, and PSAD. These data were obtained from the medical records of patients that underwent prostate biopsy from January 2008 to December 2013 in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. The inclusion criteria were patients with LUTS, PSA level >4 ng/ml, abnormal DRE, IPSS score >7, and age >45 years old, while the exclusion criteria were patients who underwent transperineal prostate biopsy or had been biopsied previously. The indications for prostate biopsy in our hospital were PSA level >4 ng/ml or abnormal DRE. All patients underwent 6–12-core biopsy using an 18-gauge needle with a spring-loaded biopsy gun (Bard Magnum). PV was calculated by measuring height (H), width (W), and length (L) of the prostate on TRUS and the formula would be: V= 0.52 x H x W x L. Collected data were further analyzed. Although this study was conducted in a single institution, we believe it is representative of Indonesian men because the samples are recruited from several provinces in Indonesia, since Cipto Mangunkusumo Hospital is a national referral hospital in Indonesia.\n\nThe study analysis was performed with SPSS version 20.0. We calculated the statistical analysis using Mann-Whitney test and unpaired t-test for the quantitative variables (age, PV, PSA and PSAD). In addition, Chi-square was used for statistical significance for qualitative variables. P <0.05 was considered as a significant result. Receiver operating characteristic (ROC) curve analysis was also conducted to determine the cut-off point and the optimum specificity and sensitivity for each variable.\n\n\nResults\n\nA total of 1232 patients who underwent prostate biopsy were retrospectively analyzed. Among those patients, 413 patients had positive biopsy results (diagnosed with PCa) and 819 patients had negative biopsy result (diagnosed with benign prostate hyperplasia (BPH) or prostatitis). The characteristics of patients in this study can be viewed in Table 1.\n\nNegative biopsy results were diagnosed as benign prostate hyperplasia and/or prostatitis while positive biopsy results were diagnosed as prostate adenocarcinoma.\n\nPSA, prostate-specific antigen; PV, prostate volume; PSAD, PSA density. #Values presented are median (range); †values presented are mean ± standard deviation.\n\nCompared to the negative biopsy group, the positive biopsy group had lower median PV, while serum PSA level and age in positive biopsy group was higher than negative biopsy group (Table 1). The mean PSAD in positive biopsy result group was higher than negative biopsy results, except PSAD in patients with PSA <4 ng/ml (Table 1).\n\nBivariate analysis using the Mann-Whitney U-test showed that age (p <0.001), PSA (p <0.001), PV (p <0.001), and PSAD in patients with PSA 4–10 ng/ml (p <0.001), 10–20 ng/ml (p <0.001), and >20 ng/ml (p <0.001) group had statistical significance differences. However, PSAD in patients with PSA <4 ng/ml (p=0.368) showed no statistically significant difference using unpaired t-test.\n\nAccording to the age groups, we classified them into three groups based on previous study9. As shown in Table 2, those aged >70 years (43.4%) had the highest detection rate. In addition, the positive biopsy rate in patients with PSA >20 ng/ml was 57.2 %. Prostate volume <60 ml had higher detection rate (41.8%) compared to PV >60 ml which only has 18.2% (Table 2).\n\nNegative biopsy results were diagnosed as benign prostate hyperplasia and/or prostatitis while positive biopsy results were diagnosed as prostate adenocarcinoma.\n\nPSA, prostate-specific antigen.\n\nThe ROC curve demonstrated that PSAD was the best predictor factor among those variables (age, PSA, and PV) in TRUS-guided prostate biopsy (Figure 1 and Table 3). The area under the curve (AUC) for PSAD (84.5%) and PSA (78.7%), while the AUC for PV and age were 62.4% and 61.2%, respectively. PSAD and PSA were thus considered better predictors for identifying PCa than PV and age. This study also conducted ROC curve analysis for PSAD in stratified PSA level (Table 3).\n\nReceiver operating characteristic curve for (A) prostate-specific antigen (PSA), PSA density (PSAD), and age, and (B) prostate volume.\n\nAUC, area under the curve; PSA, prostate-specific antigen; PV, prostate volume; PSAD, PSA density.\n\nThe optimal cut-off point of PSAD in the PSA group of 4–10 ng/ml was 0.17 ng/ml and its sensitivity and specificity were 75% and 66.5%, respectively. The cut-off point of PSA level was 32.5 ng/ml, giving a sensitivity and specificity for PSA of 62.0% and 87.1%, respectively. Prostate volume and age had lower optimum sensitivity and specificity compared to PSAD and PSA. (Table 3) This study divided PSAD variables into four groups based on PSA level (Table 3).\n\n\nDiscussion\n\nRashid et al. stated that TRUS-guided biopsy is recommended as the procedure of choice for early detection of PCa. Even though the elevation of PSA and the presence of an abnormal DRE are associated with PCa, those kind of examinations are not appropriate as a standard predictor9.\n\nIn our study, 33.5% of the 1,232 patients examined had prostate cancer based on the biopsy result of histopathology examination. The overall detection rate (33.5%) of PCa in this study was much lower than in Pakistan (50.3%)10. Na et al. and Presti et al. also showed differences compared to earlier Chinese and American studies; they reported that the detection rates were 47% and 42%, respectively11,12.\n\nThe same results in positive biopsy rate are also found in other studies (Table 4). In Pakistan, Rashid et al. showed that 48.8% of patients performing TRUS guided biopsy were detected as PCa9. In India, Prakash et al. found that 35.2% of PCa were diagnosed from prostate biopsy13. Tang et al. in China revealed that 36.4% of patients were positive for PCa after performing prostate biopsy, but this study only investigated PSA levels ranging from 10–50 ng/ml14. Study from Germany had a lower detection rate than current study, at only 23.5%15. The low detection rate in this study could be caused by inadequate sampling. In larger prostate volumes, the possibility of error sampling is higher than in smaller prostate volume. Hence, the number of cores should be adjusted based on the prostate volume.\n\nPSA, prostate-specific antigen.\n\nIn this study, the median age of PCa patients is 68 years old (range from 35–89 years) and those who were >70 years had the highest positive biopsy rate. This result is supported by other studies in Indonesia and Pakistan2,10,16. Umbas et al. revealed that patients with PCa in Indonesia were predominantly those >70 years during 1995–20042. In addition, Ariani et al. also reported that the overall age of the PCa patients in their study was 67.12 years16. The previous two studies mentioned that the rate in Indonesia was not significantly different compared to studies conducted in Nepal Senegal, Pakistan and Taiwan (63.6, 65.5, 65.7, and 67.7 years, respectively)9,17–19. However, other studies from China (Na et al. and Wu et al.) showed a slightly higher overall age compared to the current study, with mean ages of 71.24 and 73.4 years, respectively11,20. In addition, the mean age of a group in Korea was found to be lower (63.8 years old) than that in the current study, but its result cannot be compared because the study use tPSA level less than 10 ng/ml21.\n\nAge is one of many risk factors affecting PCa because the incidence of the disease increases along with aging. Prostate cancer is mostly (in about 75% of cases) diagnosed after 65 years of age. There only 2% of all PCa cases were diagnosed in males <50 years19.\n\nThe highest positive biopsy rate in PSA group within this study belongs to patients with PSA values >20 ng/ml. This result suggests that severe elevation of PSA will increase the probability of PCa. Prakash et al. and Rashid et al. showed that 73.8–74.6% of patients with PSA >20 ng/ml that underwent TRUS-guided biopsy were diagnosed with PCa10,13. Another study by Barakazi et al. revealed a similar result to the current study22.\n\nThis study shows that patients with PSA level 4–10 ng/ml and 10–20 ng/ml had lower detection rate compared to those with PSA level below 4 ng/ml. Prakash et al. also generated similar results to those of our study, showing that PCa in patients with PSA level <4 ng/ml was could be detected more easily compared to patients with PSA levels of 4–10 ng/ml and 10–20 ng/ml, with the addition of DRE examination13. These results suggested that DRE may still have an important role in assisting or predicting PCa in suspicious patients23. Previous studies conducted in Taiwan and The Netherlands also revealed that DRE is an independent predictor of PCa19,24.\n\nIn this study, the lowest detection rate of PCa is in those with a PSA level of 4–10 ng/ml. Therefore, PSA alone is not an effective screening method to diagnose PCa. PSA level alone could cause the high rate of false positive or negative biopsy rate in patients that underwent TRUS-guided prostate biopsy. PSA is a protein produced by the prostatic epithelium. It is organ-specific, but not cancer-specific. PSA can be manipulated by other disorders of the prostate, such as prostatitis, urinary retention, and BPH. Research into a large population regarding PSA screening in ERSPC revealed that PSA screening was able to reduce a number of 20% mortality in patients with PCa. Another study reported that PSA had no benefit in reducing mortality in PCa. PSA alone is not optimal to determine the risk of PCa11. Other factors such as DRE, TRUS to asses PV, and PSAD must be considered when performing prostate biopsy24.\n\nThe role of PV as a predictor factor is still being debated. In this study, the mean PV in patients with PCa is much lower than in those with negative biopsy results. A previous study in Indonesia conducted by Ariani et al. produced the same result as our study16. The study revealed that the incidence of PCa increased with decreasing PV16. Wu et al. reported that lower PV was able to indicate a higher risk of PCa detection20. Tang et al. reported that decreasing PV was associated with higher risk of PCa in patient undergoing TRUS biopsy14. The high rate of negative biopsies resulting due to larger PV might be due to more sampling errors in TRUS biopsy. Therefore, assessing more cores from larger prostates could increase detection rate and reduce bias5,14,25. Pietzak et al. revealed that patients with larger PV (50 ml) who had prior negative biopsy require multiple biopsies to increase the chance of gaining a positive biopsy26.\n\nThis study revealed that PSAD had the best predictor in detecting PCa. A study from China showed a similar ROC cut-off point (0.2 ng/ml/ml) and AUC (0.664) in intermediate PSA level (4–10 ng/ml) to this study27. The previous study in Indonesia also reported that the optimum cut-off point was 0.19 in patients with an intermediate PSA level28. The cut-off point of PSAD is similar in our study and is higher compared to the reference values of Western countries (0.15 ng/ml/ml) for prostate biopsy29. This examination could be used to reduce unnecessary biopsy because the specificity of PSAD was much higher than specificity of PSA level 4 ng/ml (as indicated for prostate biopsy) in this study.\n\nThe limitation of our study is that the TRUS-guided biopsy is of limited value. It can only show small number of malignancies. The use of contrast agent combined with TRUS-guided prostate biopsy can increase the accuracy of prostate cancer diagnosis29. We also noticed that according to Prakash et al., the number of core biopsies have to be increased up to 16 cores in order to significantly increase the rate of prostate cancer detection13.\n\nAnother limitation of this study is that it is a retrospective study conducted in a single institution. In this study, PSA level was not defined adequately, therefore the cut-off point and mean variables were difficult to obtain. Although this study is conducted in a single institution, it can represent the races from Indonesian men because the samples are recruited from several provinces in Indonesia.\n\n\nConclusion\n\nIn conclusion, the overall detection rate of PCa by biopsy in our study is 33.5%, much lower than other countries in Asia. The incidence of PCa increased with higher PSA level, older age and lower PV. Utilization of PSAD 0.17 ng/ml/ml as a cut-off point in patients with PSA level between 4–10 ng/ml is recommended to improve PCa detection in Indonesian men.\n\n\nData availability\n\nHarvard Dataverse Repository: Final Data Biopsy RSCM 2008–2013. https://doi.org/10.7910/DVN/JNNECM30.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nFerlay J, Soerjomataram I, Ervik M, et al.: Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11. [Internet] Lyon, France: International Agency for Research on Cancer; 2012; [cited 2013 1Sept 2014]. Reference Source\n\nUmbas R: Characteristics and management of prostate cancer in Jakarta over ten years period. Ind J Surgery. 2005; 33(4): 107–14.\n\nUmbas R, Mochtar C, Rahardjo H: Current Status of Prostate Cancer in Asia. Indonesian Journal of Cancer. 2011; 5(1): 21–4. Reference Source\n\nHoffman RM: Clinical practice. Screening for prostate cancer. N Engl J Med. 2011; 365(21): 2013–9. PubMed Abstract \| Publisher Full Text\n\nTeo JK, Poh BK, Ng FC, et al.: Detection rate of prostate cancer on the basis of the vienna nomogram: a singapore study. Korean J Urol. 2014; 55(4): 245–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDraisma G, Etzioni R, Tsodikov A, et al.: Lead time and overdiagnosis in prostate-specific antigen screening: importance of methods and context. J Natl Cancer Inst. 2009; 101(6): 374–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLundström KJ, Drevin L, Carlsson S, et al.: Nationwide population based study of infections after transrectal ultrasound guided prostate biopsy. J Urol. 2014; 192(4): 1116–22. PubMed Abstract \| Publisher Full Text\n\nAnastasiadis A, Zapala L, Cordeiro E, et al.: Complications of prostate biopsy. Expert Rev Anticancer Ther. 2013; 13(7): 829–37. PubMed Abstract \| Publisher Full Text\n\nRashid R, Mubarak M, Kazi J: Frequency of Adenocarcinoma in Transrectal Ultrasound-guided Prostate Needle Biopsies in Men with clinical suspicion of Prostate Cancer and Raised Serum Prostate Specific Antigen. Middle East J Cancer. 2013; 4(2): 73–8. Reference Source\n\nKash DP, Lal M, Hashmi AH, et al.: Utility of digital rectal examination, serum prostate specific antigen, and transrectal ultrasound in the detection of prostate cancer: a developing country perspective. Asian Pac J Cancer Prev. 2014; 15(7): 3087–91. PubMed Abstract \| Publisher Full Text\n\nNa R, Jiang H, Kim ST, et al.: Outcomes and trends of prostate biopsy for prostate cancer in Chinese men from 2003 to 2011. PLoS One. 2012; 7(11): e49914. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPresti JC Jr, Chang JJ, Bhargava V, et al.: The optimal systematic prostate biopsy scheme should include 8 rather than 6 biopsies: results of a prospective clinical trial. J Urol. 2000; 163(1): 163–6; discussion 166–7. PubMed Abstract \| Publisher Full Text\n\nPrakash VS, Mohan GC, Krishnaiah SV, et al.: Ten-core versus 16-core transrectal ultrasonography guided prostate biopsy for detection of prostatic carcinoma: a prospective comparative study in Indian population. Prostate Int. 2013; 1(4): 163–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTang P, Jin XL, Uhlman M, et al.: Prostate volume as an independent predictor of prostate cancer in men with PSA of 10-50 ng ml-1. Asian J Androl. 2013; 15(3): 409–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuboldt HJ, Bex A, Swoboda A, et al.: Early detection of prostate cancer in Germany: a study using digital rectal examination and 4.0 ng/ml prostate-specific antigen as cutoff. Eur Urol. 2001; 39(2): 131–7. PubMed Abstract \| Publisher Full Text\n\nAriani D, Umbas R: The Role of Prostate Volume and Serum PSA for Detecting Prostate Cancer in LUTS Patients with Normal DRE. Indonesian Journal of Cancer. 2011; 5(2): 88–93.\n\nBelbase NP, Agrawal CS, Pokharel PK, et al.: Prostate cancer screening in a healthy population cohort in eastern Nepal: an explanatory trial study. Asian Pac J Cancer Prev. 2013; 14(5): 2835–8. PubMed Abstract \| Publisher Full Text\n\nNiang L, Kouka CN, Jalloh M, et al.: Screening for Prostate Cancer by Digital Rectal Examination and PSA Determination in Senegal. ISRN Oncol. 2011; 2011: 943704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang JC, Huan SK, Kuo JR, et al.: A multivariable logistic regression equation to evaluate prostate cancer. J Formos Med Assoc. 2011; 110(11): 695–700. PubMed Abstract \| Publisher Full Text\n\nWu YS, Na R, Xu JF, et al.: The influence of prostate volume on cancer detection in the Chinese population. Asian J Androl. 2014; 16(3): 482–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhn JH, Lee JZ, Chung MK, et al.: Nomogram for prediction of prostate cancer with serum prostate specific antigen less than 10 ng/mL. J Korean Med Sci. 2014; 29(3): 338–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarakzai M, Mubarak M, Kazi J: Histopathological lesions in transrectal ultrasound guided biopsies of prostate in patients with raised serum prostate specific antigen: A preliminary report. Nephro-Urol Mon. 2011; 3(3): 186–90. Reference Source\n\nGosselaar C, Roobol MJ, Roemeling S, et al.: The role of the digital rectal examination in subsequent screening visits in the European randomized study of screening for prostate cancer (ERSPC), Rotterdam. Eur Urol. 2008; 54(3): 581–8. PubMed Abstract \| Publisher Full Text\n\nRoobol MJ, van Vugt HA, Loeb S, et al.: Prediction of prostate cancer risk: the role of prostate volume and digital rectal examination in the ERSPC risk calculators. Eur Urol. 2012; 61(3): 577–83. PubMed Abstract \| Publisher Full Text\n\nBasillote JB, Armenakas NA, Hochberg DA, et al.: Influence of prostate volume in the detection of prostate cancer. Urology. 2003; 61(1): 167–71. PubMed Abstract \| Publisher Full Text\n\nPietzak EJ 3rd, Kabarriti AE, Mucksavage P, et al.: The presence of high-grade prostatic intraepithelial neoplasia or atypia on prostate biopsy does not adversely affect prostatectomy outcomes for patients otherwise eligible for active surveillance. Urology. 2014; 84(6): 1442–7. PubMed Abstract \| Publisher Full Text\n\nZhao R, Huang Y, Cheng G, et al.: Developing a follow-up strategy for patients with PSA ranging from 4 to 10 ng/ml via a new model to reduce unnecessary prostate biopsies. PLoS One. 2014; 9(9): e106933. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMochtar CA, Rahardjo D, Umbas R: A higher PSA-density cut-off level in patients with intermediate PSA values for the early detection of prostate cancer. Gan To Kagaku Ryoho. 2000; 27 Suppl 2: 514–22. PubMed Abstract\n\nHwang SI, Lee HJ: The future perspectives in transrectal prostate ultrasound guided biopsy. Prostate Int. 2014; 2(4): 153–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPutra PCA: Final Data Biopsy RSCM 2008-2013. Harvard Dataverse, V2. 2019. http://www.doi.org/10.7910/DVN/JNNECM" }	[ { "id": "50755", "date": "04 Jul 2019", "name": "Sebastiaan Remmers", "expertise": [ "Reviewer Expertise Prostate cancer research", "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 paper evaluates predictors for a positive systematic biopsy. These predictors included PSA, age, prostate volume, and PSA density (PSAD). The authors concluded that PSAD was the best predictor for a positive biopsy based on ROC analysis. The authors have done great work by including 1232 patients. The manuscript is nicely written. There are some considerations before accepting this manuscript for indexing:\nWhy are men with normal DRE not included? If you only include men with abnormal DRE you will make a high risk cohort and you will miss tumour located on the ventral side of the prostate.\n\nIn the method section, the authors describe that inclusion criteria were age >45 years and PSA >4 ng/ml. However, in Table 1 the range for men with positive biopsy was 35-89 and for men with negative biopsy 40-83. In addition, the range of PSA is 0.8 – 7740 ng/ml. A more proper way of reporting is by providing the median and the interquartile range. Figure 1 is composed of a section A and B. However, I do not see the added value of separating prostate volume from the rest of the predictors. The methodology is not sufficient to conclude that PSAD is the best predictor. Multiple testing will lead to biased results. The best methodology is to make a logistic model in which age and PSAD are used as predictor. Since PSA and prostate volume are combined into PSAD, it is not recommended to use both PSAD, and PSA and prostate volume in one model. On the basis of this model, it is possible to calculate an AUC and to make an individual prediction for a men.\n\nBefore an accurate message can be given, it is first important to take the previous considerations into account. The results of an Indonesian cohort will be of interest for both researchers and urologists.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-875
https://f1000research.com/articles/7-1923/v1	11 Dec 18	{ "type": "Systematic Review", "title": "A systematic review and critical evaluation of immunohistochemical associations in hidradenitis suppurativa", "authors": [ "John W. Frew", "Jason E. Hawkes", "James G. Krueger", "Jason E. Hawkes", "James G. Krueger" ], "abstract": "Background: Hidradenitis suppurativa (HS) is a chronic inflammatory disease with significant morbidity and impact on quality of life. Our understanding of the pathophysiology is incomplete, impairing efforts to develop novel therapeutic targets. Immunohistochemistry studies have produced conflicting results and no systematic evaluation of study methods and results has been undertaken to date. Methods: This systematic review aimed to collate and describe all reports of immunohistochemical staining in HS. This systematic review was registered with PROSPERO and conducted in line with the PRISMA reporting guidelines. Potential bias was assessed using the NIH Criteria and antibodies used across various studies were tabulated and compared. Results: A total of 22 articles were identified describing results from 494 HS patients and 168 controls. 87 unique immunohistochemical targets were identified. The overall quality of studies was sub-optimal with staining intensity confounded by active treatment. Conflicting data was identified and able to be reconciled through critical evaluation of the study methodology. Conclusions: Keratinocyte hyperplasia with loss of cytokeratin markers co-localizes with inflammation comprising of dendritic Cells, T-lymphocytes and macrophages, which are known to play central roles in inflammation in HS. Primary follicular occlusion as a pathogenic paradigm and the principal driver of HS is not consistent with the findings of this review. Inflammation as a primary driver of disease with secondary hyperkeratosis and follicular occlusion is more consistent with the current published data.", "keywords": [ "Hidradenitis Suppurativa", "Cytokeratin", "Immunohistochemistry", "Pathogenesis", "Inflammation", "Follicular Occlusion" ], "content": "Introduction\n\nHidradenitis suppurativa (HS) is a chronic inflammatory disease, the exact pathophysiology of which remains incompletely defined1. Numerous inflammatory mediators including TNF-α2, IL-172,3, IL-324 and IL-36 subtypes5,6 have been implicated in the disease. However, there is an incomplete understanding of the source and triggers of these mediators and how they sustain the chronic inflammation that characterizes this disease1,2. The pathogenic paradigm of HS has evolved dramatically since the first description by Velpau in 18397. First thought of as an apocrinitis of infectious aetiology, it is now considered a disorder of follicular occlusion and more recently an inflammatory disease characterised by a keratinocyte mediated inflammatory response6. However, the variable response to topical, systemic and biologic therapies in HS8 indicate our understanding of disease pathophysiology is incomplete when compared to other cutaneous inflammatory diseases such as psoriasis9 and atopic dermatitis10. Existing studies examining the histology and immunohistochemical profiling of HS tissues represent conflicting results, for example in the degree of dermal dendritic cell infiltration11,12 and the production of TNF-alpha in the follicular unit13,14. These results may be influenced by heterogeneous sampling methods, laboratory processing methods and data analysis15. An additional complicating factor is that clinical comorbidities which are strongly associated with disease activity in HS, such as obesity16, diabetes17, inflammatory bowel disease18, and smoking19 also impact inflammatory cell activity in the skin18,20–22. Hence it remains unclear whether the presence or absence of these conditions may confound the findings of immunohistochemical studies in HS15,23 and whether clinical stratification of patients is required to identify distinct pathogenic pathways, which may be amenable to pharmacological intervention. This variability across studies makes comparing data problematic. To date no systematic analysis of immunohistochemical studies has been undertaken to compare results, methodology and analytical techniques.\n\n\nObjectives\n\nThe objectives of this systematic review are:\n\n1) To collate and describe all published reports of immunohistochemical studies in HS\n\n2) To critically evaluate the sampling, laboratory and analysis techniques used in each study to determine if comparisons can be made across studies.\n\n\nMethods\n\nThis systematic review was registered with PROSPERO24 (Registration number CRD42018104763) and was conducted in line with the PRISMA25. The STROBE statement26 was used to assess the observational studies included in this study.\n\nInformation Sources for this review encompassed Pubmed (1946-July 1 2018), Scopus (2004- July 1 2018) and Web of Science (1990-July 1 2018) as shown in Figure 1. Search strategy is presented as Table 1.\n\nEligibility criteria for this review included cohort studies, case-control studies and other observational studies with no restrictions of patient age, sex, ethnicity or language of publication. Eligible studies included those reporting the results of immunohistochemical findings in HS. Studies deemed not eligible included articles which provided no new data, only a review or summary of previously published data.\n\nData collection was performed independently by 2 authors (JWF & JEH), with any disagreements regarding inclusion of citations being referred to a third author (JGK) for mediation. Information was collected using a standardized data collection form (available as Extended data27) with the principal outcomes of interest being the immunohistochemical stain of interest, the site and rated intensity of staining (as described by authors), and comparison with perilesional/ unaffected/ control tissue. If data from individual patients was not available then the aggregate data was collected.\n\nPotential sources of bias in the identified studies are acknowledged including the small size of patient cohorts, the variability in sampling and laboratory techniques, antibodies published and reactants used. Therefore these variables (where available) were collated to assess the heterogeneity of studies. Bias was also assessed using the NIH quality assessment tool for observational studies28.\n\n\nResults\n\nA total of 425 non-duplicated citations were identified in the literature review (Figure 1). 398 of these articles were removed upon review of titles and abstracts against the pre-defined eligibility criteria. Full text review of the remaining 27 articles excluded 5 articles providing no new data. The remaining 22 studies4–6,11–14,29–43 reporting the results of 494 individual HS patients and 168 control patients were used as the basis of this systematic review.\n\nThe demographics of the patients of the included studies are presented in Table 2. Of 494 HS patients, 180 were male (38.3%) and 290 female (61.7%) with 24 cases unreported. Ages ranged from 15–72 years. 47/50 (94%) of reported cases were smokers, 12/30 (40%) had a BMI >30, and there was no information pertaining to diabetes or family history of HS. Of the 200 documented biopsy sites 93 were axillae (46.5%), 69 were inguinal (34.5%), and 38 were genital (19%) (Table 3). 64 patients had Hurley staging with 7/64 (10.9%) Stage 1, 40/64 (62.5%) Stage 2 and 17/64 (26.6%) Stage 3. No individual Sartorius scores were reported. Where current treatment was reported, 6 patients (4.5%) were on adalimumab, 42 (31.6%) were untreated, 85 patients (63.9%) had treatment withheld prior to biopsy, and 357 cases were unreported. Lesional biopsies were taken from all studies, with 3 individual studies also taking perilesional biopsies6,31,41 . Age and Sex matched controls were present in 3 studies29,31,43 and results were stratified in a minority of studies. 2 studies stratified by disease severity4,12, 7 studies stratified by lesion site13,33–37,39, 5 studies stratified by treatment4,5,12,29,32, and no studies stratified by comorbidities. Analysis of immunohistochemical staining methodology varied and included quantitative analysis (3 studies)14,30,32, semi-quantitative analysis (14 studies)4,5,11–13,29,34,37–43, and the presence or absence of staining (5 studies)6,31,33,35,36. A total of 87 distinct immunohistochemical staining targets were identified (Table 4,Table 5 and Table 6).\n\nBMI= Body Mass Index mHSS= modified Hidradenitis Suppurativa Score (Sartorius Score) NR= Not Reported\n\nTable 3: Critical Evaluation of Methodology of Studies Included in This Review Key:L= Lesional, PL= Perilesional, U= Uninvolved, C= Control S=Serum, Y=Yes, N=No, NR= Not Reported, CTx-FITC =Cholera Toxin\n\nKey: + to ++++ = Degree of positive staining, - = reported negative staining, NC= No Change; ↓ Decreased, NOS= Not Otherwise Specified, *= Statistically significant result compared with healthy controls, §=Pan Cytokeratin Stain, DC= Dendritic Cells, Single K= Single keratinocytes,\n\nEpidermis. The epidermis of HS lesional tissue expressed the normal array of keratins (K) in the basal (K5, K14) and suprabasal (K1, K2e, K10) layers. Increased K6, K16 and K17 staining were increased compared to healthy controls in the suprabasal epidermis in one study30, however, K6 and K17 staining was not increased in the epidermis (only in non-keratinized portions of sinus tracts) in a second study38. Where K6 and K17 were positive in suprabasal epidermis, K17 staining was more pronounced than K6 staining30. K19 was weakly positive in acanthotic epidermis37. Ki67 staining was elevated in basal and suprabasal epidermis. Normal staining patterns of desmoplakin, plakophilin and plakoglobin were seen38. Cells staining positive for CD1a, CD206, CD207 and CD209 were seen throughout the epidermis40. CD3, CD4, CD8 and to a lesser degree CD68 positive cells demonstrated epidermotropism in sites of epidermal acanthosis30,33. CD29 and cholera toxin (double positive) staining cells were seen on the slopes of papillae of the epidermis35. hBD2 (human beta defensin) staining was decreased throughout the epidermis in two studies13,41 whilst hBD3 staining was increased throughout the suprabasal epidermis14,42, however only significantly in Hurley Stage 1 and 2 patients (p=0.045)42. hBD4 was decreased in suprabasal epidermis compared to healthy controls (p=0.001)41. Contradictory findings were seen in toll like receptor (TLR) 2 staining with an increase in the epidermis co-localizing with dendritic cells and macrophages in one study40 but suppressed in a second study41. Levels of TLR3, TLR4, TLR7, TLR9, ICAM-1, TGF-Beta and IGF-1 were only assessed by one study and all were suppressed throughout the epidermis compared with controls41. RNAase7 was increased in expression compared to healthy controls (p<0.05)42. MMP2 was positively expressed in keratinocytes throughout the epidermis13 and MMP8 in neutrophils within the epidermis43. TNF-α was highly expressed in macrophages and lymphocytes present in the epidermis, particular in the basal layers13,14 and NLRP3, MIF, S100A7, LL37/Cathelicidin and α-MSH all positive in suprabasal keratinocytes30,41. IL-6 and IL-10 were reported as suppressed compared to healthy control skin41, however, IL-36 subtypes were highly expressed in epidermal keratinocytes (more suprabasal than basal)5,6 with IL-32 also positive in the stratum granulosum4.\n\nDermis. CD1a, CD11c, CD206, CD207, CD209 and Factor XIIIa positive cells were identified in the dermis in three separate studies12,30,40, however the degree of infiltration varied. Dermal infiltrates of CD3, CD4, and CD8 positive cells, continuous with the epidermal infiltrates were a consistent feature of lesional HS dermis and were increased over controls30,33. The distribution of these cells was most pronounced in the interfollicular dermis (ie. towards the papillary slopes) and perifollicularly (ie. peri-infundibularly)30,33. CD56, CD68 and CD138 positive cells were diffusely seen throughout the dermis40. CD19 and CD20 positive pseudolymphoid follicles have been noted in other studies30,40. Single keratinocytes have also been identified in the dermis which stain with pancytokeratin markers (AE1/AE3/PKC26)36. Inflammatory cells in the dermis co-localized with TNF-α13,14, LL-37/cathelicidin29, IL-1232, IL-2332, IL-1730,32, IL-324, TLR240 and MMP843. MMP2 co-localized with macrophages and fibroblasts13. IL-36 was not identified in the dermis5,6.\n\nHair follicle. Cytokeratin staining of the follicular apparatus is consistent with normal K14, K16 and K17 staining. CD29 positive cells were identified in the infundibulum35. CD3, CD4, CD8, CD68, Factor XIIIa positive cells were seen within the outer root sheath (ORS) contiguous with dense peri-follicular inflammation in the adjacent dermis30,33. The presence of inflammatory cells co-localized with MMP213, TNF- α13,14, and LL37/cathelcidicin13,29. hBD313,42 and MIF13 also stained positive in the ORS. One conflicting study reported no change in TNF-α staining of the follicular unit13.\n\nSinus tracts. Staining patterns differed between superficial keratinized sinus tracts and deeper, inflamed non-keratinized sinus tracts. Normal epidermal cytokeratin staining was seen in the keratinized superficial portion of sinus tracts including K1, K10, K1436–38. Ki67 was elevated and CD29 positive cells were also identified in sinus tracts35. Ki67 stained in both keratinized and non-keratinised portions of the sinus tract38. K19 staining was absent in keratinized portions of sinus tracts37. In deeper, inflamed, non-keratinized portions of the sinus tracts, K16, K17 and K19 were positive, with loss of K1, K10 and adhesions molecules including DG1 (desmoglein 1)and DSC1 (desmocollin 1)37,38. Apocrine gland nuclei stained weakly positive for estrogen receptor39 and androgen receptor39, and these results were reported as no different from control specimens39. Lysozyme staining of apocrine glands was seen in cases of vulval HS only34.\n\nImmunohistochemistry methods. The list of antibodies used for IHC staining is presented in Table 6. Consistent antibodies were used for CD1a; CD20 and tryptase staining, whilst different antibodies were used for other staining targets. Antibodies used were not described in two studies34,36.\n\nAssessment of Bias. The result of bias assessment using NIH criteria is presented in Table 7. All 22 articles clearly stated the research question of interest with well-defined study populations. The application of inclusion and exclusion criteria, or the calculation of sample size, or effect estimates were not described in any study. Exposures (ie. the presence of disease) were established and measured in all studies prior to the outcome measures (IHC staining) being assessed and the disease was established for such a time that a relationship between exposure and outcome would be identified if one existed. Different levels of exposure (severity of disease) was taken into account in only two studies4,12 and was consistently measured using Hurley staging across all studies. No articles accounted for all possible confounding variables such as obesity, diabetes, family history or smoking status (Table 3).\n\nKey: Y = Yes; N= No, NR= Not Reported N/A = Not Applicable\n\n\nDiscussion\n\nThe overall quality of data in this systematic review was sub-optimal with poor correction for potential confounding factors with only two of the 22 studies using objective measurement systems for IHC staining intensity4,12. The proportion of smokers was elevated (94%) compared to the rates of smoking in the HS population at large (70–89%)45. A number of studies (17/22) did not stratify results by treatment therefore there is a risk that staining intensity of pro-inflammatory mediators may be reduced due to concomitant treatment at the time of biopsy. The use of de-paraffinized tissue in retrospective studies30,33,34 can lead to false negatives in IHC dependent upon the preparation method of the original sample and the de-paraffinization process15. Hence there are factors in the population studied in this review which may bring into question the reliability of staining quantification. However, the presence or absence of IHC staining, particularly when confirmed in multiple studies is still considered reliable despite the risks of bias.\n\nConflicting results were identified in dermal CD1a staining12,30,40, dermal CK19 staining36–38, Epidermal TLR2 staining40,41 and TNF alpha staining in the follicular infundibulum13,14. Regarding CD1a staining, two of the studies reported only a mild dermal infiltrate of CD1a positive cells30,40, with a third study demonstrating a significant infiltration of these cells12. This third study clearly documented all treatment was withheld 3 weeks prior to the biopsies being taken12, whereas there is no description in the other two articles regarding the discontinuation or ongoing use of treatments30,40. Therefore, with the possibility of partially treated disease, an artificial reduction in the number of dermal dendritic cells is a possibility as treatment for HS (such as adalimumab) has been demonstrated to effectively reduce the infiltration of dendritic cells in vivo12. Similarly, studies examining TNF-alpha staining also differed in their stratification of patient based upon active treatment13,14. Significant reductions in TNF alpha staining were seen in the study with no documentation of treatment cessation14 when compared to the one study with clear documentation that all patients had treatment ceased prior to biopsy13. K19 staining was reported negative in all areas of the sinus tracts in one study37, whereas two additional studies36,38 described positive K19 staining in sinus tracts (one study non-specifically36 and the second in the deep inflamed, non-keratinized epithelium of the tract38). The difference between these staining patterns may be explained by the presence of inflammation. Kurzen et al.38 described the presence of K19 staining in non-keratinized epithelium of the deep sinus tracts only when associated with inflammation (Type 3 epithelia), staining was negative when no inflammation was present (Type 2 epithelia)38. Kurokawa et al. did not differentiate between inflamed and non-inflamed non-keratinized epithelium in their study37, and noted that the lesser degree of inflammation seen histologically may explain their differing results in comparison to Kurzen’s study37.\n\nIHC staining, in particular co-staining with cellular markers and cytokines has enabled the localization of inflammatory mediators in order to ascertain the functional aspects of infiltrating inflammatory cells in HS, particularly highlighting the strong Th17 polarity of inflammation in HS3. A schematic representation of the pathogenesis of HS based upon the findings of this review is presented in Figure 2. This highlights the inter-relationship between inflammation and hyperkeratinization. Localization of TNF-α13, IL-1232, IL-2332 and IL-324, TLR240, MMP213, MMP843 and LL-37/cathelicidin14,29 production to infiltrating dermal macrophages and lymphocytes as well as localization of IL-36 subtypes5, LL-37/cathelicidin14,29, IL-1β32 and IL-2232 to keratinocytes illustrate the feed forward mechanisms similar to those seen in psoriasis9 and atopic dermatitis10 which likely contribute to persistent inflammation in HS. Rather than keratinocytes being innocent bystanders, these IHC findings demonstrate the central role keratinocytes play as producers of key inflammatory mediators as well as mediators of products (such as TGF-β and ICAM)41 that may contribute to fibroblast dysregulation and hypertrophic scarring46. A remaining unanswered question includes the temporal relationship between keratinocyte hyperproliferation and the activation of inflammatory cells infiltrating the dermis and epidermis in HS.\n\nImmunological ‘priming’ occurs due to the contribution of adipose tissue, genetic susceptibility, smoking-related inflammatory mediators and obesity related pro-inflammatory signals and the composition of the microbiome. Increased activity of cDC1, cDC2 and T cells lead to both keratinocyte hyperplasia via the actions of IL-12 and IL-23, as well as a Th17 predominant immune response. Alterations of antimicrobial peptides (AMP’s) also occur throughout the epidermis. IHC staining localize Langerhan cells and activated dendritic cells to the epidermis and the dermo-epidermal junction. A population of epidermotropic CD8 T cells are also present. IHC staining indicates a mixed inflammatory infiltrate in the dermis, with contributions from Dendritic cells, B cells, T cells and plasma cells. Within sinus tracts, adhesion molecules are preserved, but inflammation in associated with non-keratinised sinus tracts leads to a loss of K19. The development of scarring and sinus tracts is associated with MMP2, ICAM-1 and TGF-Beta, with possible augmentation of ICAM-1 and TGF-B signaling via specific components of the microbiome. TNF-a, PGE2 and CXCL2 then lead to additional feed forward mechanisms perpetuating the inflammatory cycle.\n\nThe current pathophysiological paradigm of HS is one of follicular infundibular occlusion leading to follicle rupture and a resultant inflammatory cascade1. This paradigm was based on the pivotal work of Shelley and Cahn in 195547, whom demonstrated the induction of HS after application of belladonna impregnated tape to manually epilated axillae of 12 men. Only 3 of the 12 men developed the lesions described, and infection from the manual epilation procedure could not be excluded as a cause of the lesions, but this study enabled the paradigm to slowly shift away from one of apocrinitis, which had been in place since the original descriptions of the disease7. Detailed descriptions of infundibular hyperkeratosis (also termed poral occlusion) were made by Jemec et al.7 and demonstrated the secondary involvement of apocrinitis in HS lesions. Jemec noted that poral occlusion was seen to occur alongside inflammation, but there was no suggestion of causation in one direction or another7.\n\nAlthough individual cases of epidermal hyperkeratosis in the absence of inflammation are noted7,33, these cases are established or chronic lesions associated with significant fibrosis which is documented to be associated with reduce inflammatory infiltrate7,33. A consistent finding in all studies of this review is the co-localization of infundibular ORS keratinocyte hyperplasia with CD3, CD4, CD8 and CD68 positive inflammatory cells expressing TNF-α, IL-12, IL-23 and IL-324,13,32,40,43. K19 is also documented as positive in the infundibulum suggesting keratinocyte hyperplasia36–38. However, it remains unclear whether keratinocyte hyperplasia induces the inflammatory cascade or if the inflammatory cascade induces the keratinocyte hyperplasia. The presence of inflammation in clinically normal, peri-lesional HS skin is well documented4,30,33 implying the existence of a pre-clinical inflammation preceding symptoms of follicular occlusion. This is consistent with recent findings in acne pathogenesis that suggest that inflammation precede follicular hyperkeratosis and development of microcomedones48 and is also pivotal in the ongoing development of nodulocystic acne and acne scars49. This pre-clinical inflammation is also consistent with the pathogenic paradigm in psoriasis and atopic dermatitis9,10 with inflammation driving epidermal hyperkeratosis and alterations in keratinocyte maturation, consistent with the spongiform infundibulfolliculitis seen in established lesions of HS50. Our disparate findings in K19 staining in deep non-keratinized sinus tract epithelia with and without inflammation37,38 also fit with this paradigm. In contrast, findings which would hold consistency with the current follicular occlusion paradigm would include infundibular occlusion preceding the development of inflammation, as well as alterations to desmosomal and hemidesmosomal proteins which would allow for rupture of the occluded follicles in order to drive the development of dermal inflammation and sinus tract formation. Although Danby et al.51 reports reduced PAS positivity in the basement membrane zone at the sebo-follicular junction associated with inflammation in HS, it is likely that the reduced basement membrane integrity is secondary to inflammation and release of TGF-β and MMP252 (cytokines known to be altered in HS lesional skin and consistent with an abnormal wound healing response) rather than the follicular rupture being the primary driver of inflammation.\n\nA more consistent hypothesis which accounts for the observed results of this review would be that of subclinical inflammation (due to a variety of triggers and immunological primers as illustrated in Figure 2) driving keratinocyte proliferation in the interfollicular epidermis and the follicular ORS, with follicular occlusion being a secondary phenomenon (mediated by TLR2 and IL-1α as documented in the development of comedones)53. The development of sinus tracts and hypertrophic scarring may also be mediated by the keratinocyte inflammatory response given the alterations in important wound healing mediators including TGF-β, ICAM-1 and comparisons by other authors of an altered wound healing response8 in HS. This comparison would be appropriate given the high levels of dermal MMP213 and MMP843; the loss of keratinocyte maturation markers (K2e, K10, K19)36–38 adhesion molecules (DG1 and DCN2)38 in the non keratinized inflamed epithelium of the deep dermis; suppressed levels of ICAM-141 (seen impaired wound healing54) and TGF-β41 which leads to the dysregulation of TGF- β receptor ratio on fibroblasts which is linked with the development of hypertrophic scarring46,54 seen in HS. These alterations to keratinocyte maturation are reminiscent of epithelial mesenchymal transition (EMT)52 which may also explain the presence of free keratinocytes in the dermis in established lesions of HS7,36. Indeed, as ICAM-1 is up-regulated by pro-inflammatory mediators54, the low level of ICAM-1 noted appears paradoxical, however specific bacteria (including Porphyromonas species) which have been associated with HS44,55 can suppress ICAM-1 production as an immune evasion strategy56. This implies that exogenous triggers (possibly including bacterial stimuli) can be a common cause for the initial inflammatory cascade as well as the development of tunneling and hypertrophic scarring in HS.\n\n\nConclusions\n\nThis systematic review of immunohistochemical staining of lesions in HS has highlighted the heterogeneity of studies and the methodological issues, which bring into question some of the results of IHC staining in HS lesions. The design of studies and variable reporting of potential confounding factors (such as ongoing or previous treatments) makes it impossible to compare staining intensity across studies. The results of existing studies suggest a florid inflammatory reaction comprising of T-lymphocytes, macrophages and dendritic cells with a strong Th-17 signature along with a keratinocyte mediated IL-36 inflammatory loop associated with keratinocyte hyperproliferation. The follicular occlusion paradigm as a primary driver of HS is not consistent with the findings of this review and other histological and cytokine studies and inflammation as a primary driver of disease with secondary hyperkeratosis and occlusion is a more consistent hypothesis.\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\nOSF: Extended data. Data collection sheet. https://doi.org/10.17605/OSF.IO/2JKPW27\n\nLicence: CC0 1.0 Universal\n\nOSF: PRISMA Checklist for ‘A systematic review and critical evaluation of immunohistochemical associations in hidradenitis suppurativa’. https://doi.org/10.17605/OSF.IO/2JKPW27\n\nLicence: CC0 1.0 Universal", "appendix": "Grant information\n\nSupported in part by a grant from the National Center for Advancing Translational Sciences (NCATS) [UL1 TR001866], National Institutes of Health (NIH) Clinical and Translational Science Award (CTSA) program.\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\nHoffman LK, Ghias MH, Lowes MA: Pathophysiology of hidradenitis suppurativa. Semin Cutan Med Surg. 2017; 36(2): 47–54. PubMed Abstract \| Publisher Full Text\n\nMelnik BC, John SM, Chen W, et al.: T helper 17 cell/regulatory T-cell imbalance in hidradenitis suppurativa/acne inversa: the link to hair follicle dissection, obesity, smoking and autoimmune comorbidities. Br J Dermatol. 2018; 179(2): 260–272. PubMed Abstract \| Publisher Full Text\n\nMoran B, Sweeney CM, Hughes R, et al.: Hidradenitis Suppurativa Is Characterized by Dysregulation of the Th17:Treg Cell Axis, Which is Corrected by Anti-TNF Therapy. J Invest Dermatol. 2017; 137(11): 2389–2395. PubMed Abstract \| Publisher Full Text\n\nThomi R, Yerly D, Yawalkar N, et al.: Interleukin-32 is highly expressed in lesions of hidradenitis suppurativa. Br J Dermatol. 2017; 177(5): 1358–1366. PubMed Abstract \| Publisher Full Text\n\nThomi R, Kakeda M, Yawalkar N, et al.: Increased expression of the interleukin-36 cytokines in lesions of hidradenitis suppurativa. J Eur Acad Dermatol Venereol. 2017; 31(12): 2091–2096. 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Exp Dermatol. 2018; 27(2): 172–177. PubMed Abstract \| Publisher Full Text\n\nLima AL, Karl I, Giner T, et al.: Keratinocytes and neutrophils are important sources of proinflammatory molecules in hidradenitis suppurativa. Br J Dermatol. 2016; 174(3): 514–521. PubMed Abstract \| Publisher Full Text\n\nWolk K, Wenzel J, Tsaousi A, et al.: Lipocalin-2 is expressed by activated granulocytes and keratinocytes in affected skin and reflects disease activity in acne inversa/hidradenitis suppurativa. British J Dermatol. 2017; 177(5): 1385–1393. PubMed Abstract \| Publisher Full Text\n\nSchlapbach C, Hanni T, Yawalkar N, et al.: Expression of the IL-23/Th17 pathway in lesions of hidradenitis suppurativa. J Am Acad Dermatol. 2011; 65(4): 790–8. PubMed Abstract \| Publisher Full Text\n\nvon Laffert M, Stadie V, Wohlrab J, et al.: Hidradenitis suppurativa/acne inversa: bilocated epithelial hyperplasia with very different sequelae. Br J Dermatol. 2011; 164(2): 367–371. PubMed Abstract \| Publisher Full Text\n\nHeller DS, Haefner HK, Hameed M, et al.: Vulvar hidradenitis suppurativa. Immunohistochemical evaluation of apocrine and eccrine involvement. J Reprod Med. 2002; 47(9): 695–700. PubMed Abstract\n\nGniadecki R, Jemec GB: Lipid raft-enriched stem cell-like keratinocytes in the epidermis, hair follicles and sinus tracts in hidradenitis suppurativa. Exp Dermatol. 2004; 13(6): 361–363. PubMed Abstract \| Publisher Full Text\n\nFismen S, Ingvarsson G, Moseng D, et al.: A clinical-pathological review of hidradenitis suppurativa: using immunohistochemistry one disease becomes two. APMIS. 2012; 120(6): 433–440. PubMed Abstract \| Publisher Full Text\n\nKurokawa I, Nishijima S, Kusomoto K, et al.: Immunohistochemical study of cytokeratins in hidradenitis suppurativa (acne inversa). J Int Med Res. 2002; 30(2): 131–136. 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PubMed Abstract \| Publisher Full Text\n\nHofmann SC, Saborowski V, Lange S, et al.: Expression of innate defense antimicrobial peptides in hidradenitis suppurativa. J Am Acad Dermatol. 2012; 66(6): 966–974. PubMed Abstract \| Publisher Full Text\n\nTsaousi A, Witte E, Witte K, et al.: MMP8 Is Increased in Lesions and Blood of Acne Inversa Patients: A Potential Link to Skin Destruction and Metabolic Alterations. Mediators Inflamm. 2016; 2016: 4097574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRing HC, Thorsen J, SAunte DM, et al.: The Follicular Skin Microbiome in Patients With Hidradenitis Suppurativa and Healthy Controls. JAMA Dermatol. 2017; 153(9): 897–905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMicheletti RG: Hidradenitis suppurativa: current views on epidemiology, pathogenesis, and pathophysiology. Semin Cutan Med Surg. 2014; 33(3 Suppl): S48–S50. PubMed Abstract \| Publisher Full Text\n\nZhu Z, Ding J, Tredget EE: The molecular basis of hypertrophic scars. Burns Trauma. 2016; 4: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShelley WB, Cahn MM: The pathogenesis of hidradenitis suppurativa in man; experimental and histologic observations. AMA Arch Derm. 1955; 72(6): 562–565. PubMed Abstract \| Publisher Full Text\n\nDel Rosso JQ, Kircik LH: The sequence of inflammation, relevant biomarkers, and the pathogenesis of acne vulgaris: what does recent research show and what does it mean to the clinician? J Drugs Dermatol. 2013; 12(8 Suppl): s109–115. PubMed Abstract\n\nHolland DB, Jeremy AH: The role of inflammation in the pathogenesis of acne and acne scarring. Semin Cutan Med Surg. 2005; 24(2): 79–83. PubMed Abstract \| Publisher Full Text\n\nBoer J, Weltvreden EF: Hidradenitis suppurativa or acne inversa. A clinicopathological study of early lesions. Br J Dermatol. 1996; 135(5): 721–5. PubMed Abstract \| Publisher Full Text\n\nDanby FW, Jemec GB, Marsch WCh, et al.: Preliminary findings suggest hidradenitis suppurativa may be due to defective follicular support. Br J Dermatol. 2013; 168(5): 1034–1039. PubMed Abstract \| Publisher Full Text\n\nKalluri R, Weinberg RA: The basics of epithelial-mesenchymal transition. J Clin Invest. 2009; 119(6): 1420–1428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSelway JL, Kurczab T, Kealey T, et al.: Toll-like receptor 2 activation and comedogenesis: implications for the pathogenesis of acne. BMC Dermatol. 2013; 13: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoivisto L, Heino J, Hakkinne L, et al.: Integrins in Wound Healing. Adv Wounds Care (New Rochelle). 2014; 3(12): 762–783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuen-Revillet H, Jais JP, Ungeheuer MN, et al.: The Microbiological Landscape of Anaerobic Infections in Hidradenitis Suppurativa: A Prospective Metagenomic Study. Clin Infec Dis. 2017; 65(2): 282–291. PubMed Abstract \| Publisher Full Text\n\nHuang GT, Kim D, Lee JK, et al.: Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms. Infect Immun. 2001; 69(3): 1364–1372. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "42315", "date": "25 Feb 2019", "name": "Gregor B. E. Jemec", "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 authors provide a systematic review of immunohistochemical studies of hidradenitis suppurativa (HS). The clearly stated objectives are:\nTo collate and describe all published reports of immunohistochemical studies in HS. To critically evaluate the sampling, laboratory and analysis techniques used in each study to determine if comparisons can be made across studies.\n\nThe review was registered with PROSPERO and conducted in line with the PRISMA. The STROBE statement was used to assess the observational studies included in the study. A PRISMA flow chart and a search strategy are provided accordingly.\nThe authors adequately discuss the confounding factors and risk of bias, which both are significant weaknesses identified in the literature by this manuscript based on limited studies.\n\nOnly 22 articles were identified describing results from 494 HS patients (average 22 pts/study) and only 168 controls. Furthermore, 87 unique immunohistochemical targets were identified adding to the scarcity of hard data. It is therefore less surprising that conflicting data were found. The authors are however able to provide a realistic analysis of the data taking these limitations into account, and, in addition, provide coherent analyses and a testable paradigm for the pathomechanisms of HS.\nThe paper provides an excellent overview of the limited number of explorative immunohistochemical studies of HS, and thus provides an important stepping-stone to further studies.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "45666", "date": "19 Mar 2019", "name": "Martin M. Okun", "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 is a thorough, thoughtful, and valuable contribution to the scientific literature on pathogenesis of hidradenitis suppurativa.\nThe principal advances of the systematic review are:\ncogently advances a reasonable hypothesis to explain decreased levels of inflammatory marker density in some studies as due to the lack of treatment interruption; links the presence of certain hyperproliferative keratin markers (K19) to the presence of concomitant inflammation collates evidence from multiple studies demonstrating the presence of keratinocyte-derived pro-inflammatory biomarkers, reinforcing the concept that keratinocytes are actively contributing to the inflammatory milieu\nThe authors advance the hypothesis that follicular occlusion is secondary to inflammation, based on:\nabsence of evidence of follicular occlusion without inflammation (though absence of evidence is not equivalent to the evidence of absence) presence of inflammation in clinically normal perilesional skin (though inflammation could be spill-over from adjacent inflamed skin) analogies with other inflammatory skin diseases presence of K19 staining only in inflamed sinus tracts (though the relevance of this observation for the pathogenesis of HS is uncertain)\nIn short, this hypothesis is plausible but the conclusion that \"primary follicular occlusion as a pathogenic paradigm and the principal driver of HS is not consistent with the findings of this review\" seems too sweeping a statement based on the information provided. The authors should consider altering this conclusion in line with the limitations of available data.\nAs a minor issue, there is an unnecessary repetition in the second sentence of the Immunohistochemistry results, epidermis section: \"Increased K6, K16 and K17 staining were increased...\"\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-1923
https://f1000research.com/articles/8-60/v1	15 Jan 19	{ "type": "Research Article", "title": "HIV prevalence correlated with circumcision prevalence and high-risk sexual behavior in India's states: an ecological study", "authors": [ "Chris R. Kenyon" ], "abstract": "Background: HIV prevalence varies between 0% and 1.6% in India's states. The factors underpinning this variation are poorly defined. Methods: We evaluated the relationship between HIV prevalence by state and a range of risk factors in the Indian 2015 National Family Health Survey. Pearson’s correlation was used to assess the relationship between HIV prevalence and each variable. The prevalence of each risk factor was compared between five high-HIV-prevalence states (>1% prevalence) and a large low-HIV-prevalence state (Uttar Pradesh; HIV prevalence, 0.06%). Results: There was an association between HIV prevalence and men's mean lifetime number of partners (r = 0.55; P = 0.001) and men reporting sex with a non-married, non-cohabiting partner (r = 0.40; P = 0.014). In general, men in high-prevalence states were less likely to be circumcised and (with the exception of Chandigarh) use condoms at last sex. In two high prevalence states (Mizoram and Nagaland), men reported a higher number of lifetime partners and a higher prevalence of multiple partners and high-risk sex in the past year. Conclusions: Variation in circumcision prevalence and sexual behavior may contribute to the large variations in HIV prevalence by state in India.", "keywords": [ "India", "HIV prevalence", "high-risk sex", "sexual network", "ecological", "circumcision" ], "content": "Introduction\n\nThere is little consensus as to whether or not differences in sexual behavior play an important role in determining the large differences in HIV prevalence between populations. Certain authors have claimed that non-behavioral factors such as differences in prevalence of herpes simplex virus 2 infection, circumcision rates and STI treatment efficacy are responsible for differences in HIV prevalence1,2. Other authors have found that HIV prevalence is associated with high-risk sexual behavior3–5. These latter studies have included ecological-level analyses. The rationale behind ecological studies is that the prevalence of sexually transmitted infections (STIs) is, to an important extent, determined by the structure of the local sex network6. This is a population-level characteristic and thus ecological studies are required to assess if correlates of this network structure are associated with HIV prevalence7. A number of studies3,5,8,9, but not all studies10, have found that correlates of network connectivity, such as rate of partner change and the prevalence of sexual partner concurrency, are positively associated with HIV prevalence. These associations have been found to be strongest when comparing ethnic groups and regions within countries3,5,8,9,11,12. Nationally representative, HIV-serolinked Demographic Health Surveys (DHS) have been a particularly useful resource for these studies3,8,11.\n\nIndia is an interesting country to test the network connectivity thesis. HIV was first detected in India in 1986 and since then a range of sources have documented higher HIV prevalence in certain North East States and to a lesser extent Karnataka, Andhra Pradesh and surrounding areas13–17. Individual-level analyses have found that well-established risk factors, such as partner number, contact with sex workers, intravenous drug usage and lack of circumcision, were associated with HIV infection13,17–20. These analyses have, however, not explained the differential HIV prevalence by state. A number of authors have argued that the higher HIV prevalence in Northeast India may be due to patterns of intravenous drug use (IVDU) in this region. Differences in contact with sex workers, patterns of migration, gender inequality and non-traditional forms of sex work, such as devadasi in Karnataka (where women are 'married' to temple deities and have sex with temple attendees), are some of the other factors that have been advanced as reasons for differences in HIV spread in India, often with little supporting evidence13,16–20. In this analysis we address this issue by assessing whether there is an ecological-state-level association between various HIV risk factors and HIV prevalence.\n\n\nMethods\n\nIndia is a federal union comprising 29 states and 7 union territories (Figure 1). We refer to these 36 entities as states.\n\nReprinted from 35 under a CC BY license, with permission from Dörrbecker, original copyright 2005.\n\n\nData source\n\nWe used the 2015 National Family Health Survey (NFHS-4) for this study. The study received ethical committee clearance for data analyses such as the one performed here. As a result, no specific ethics committee approval was necessary for this study.\n\nThe NFHS-4 used a household-based, two-stage stratified sampling approach. The first stage selected primary sampling units (PSUs) from the 2011 National Census. PSUs were villages in rural areas and census enumeration blocks in urban areas. In the second stage of the survey, 22 households were randomly selected with systematic sampling from every selected rural and urban cluster.\n\nA total of 98% of selected households were successfully interviewed. In the interviewed households, women aged 15–49 and men aged 15–54 were eligible to participate. The individual response rate for women was 97% and 92% for men. The response rates were high in all states except for Delhi and Chandigarh (Table 1). The behavioral questionnaire was administered in 17 local languages using computer-assisted personal interviewing. Further details of the survey are provided in Table 1 and the NFHS-4 report14.\n\nPrevalence of HIV and associated risk factors in 15–49-year-old women and 15–54-year-old men by state in India in 2015 (arranged according to descending HIV prevalence).\n\naAverage age in years by state.\n\nb \"90+ Partners\" – This variable refers to the percent per state reporting more than 90 lifetime sex partners (for men and women) separately, of all respondents.\n\nc Highest education level attained: Percent whose highest educational attainment is primary on no schooling.\n\nd Percent in the Poorest Wealth Quintile: this variable refers to the percent of the respondents from this region that were calculated to fall in the poorest 20% (quintile) of the nationally derived wealth band. The wealth quintiles were derived from an asset index.\n\ne Lifetime number of partners refers to the mean number of lifetime partners reported by those who reported being sexually experienced, excluding those with more than 90 partners.\n\n\nMeasures\n\nIn a random subsample of households, a dried blood spot from a finger-prick blood specimen was obtained from eligible women age 15–49 and men age 15–54 who consented to laboratory HIV testing. Respondents did not have access to the test results but were all referred to counseling and testing services in the local area. Dried blood samples were collected and subsequently tested for HIV using a Microlisa ELISA. Positive tests and a random 2% sample of negative samples were then tested with SD Bioline 1/2 ELISA (Standard Diagnostics Inc., Kyonggi-do, South Korea). Discordance between the two tests was resolved with Western Blot (BioRad). With the exception of Delhi, HIV testing rates were high in all states (median 95.9%, IQR 94.4-97.9%). Coverage was higher in women (95%) and men (90%) in rural areas as well as urban areas (women, 91%; men, 84%).\n\nThe NFHS-4 was designed to provide HIV prevalence estimates that are representative for the women (15–49 years) and men (15–54 years) for the whole country and rural and urban areas. It also oversampled certain states so as to be able to generate representative samples for 11 groups of states (Table 2). These were states that had been found to have higher HIV prevalence rates in previous surveys, as well as the Uttar Pradesh-centered group of states, which was chosen as a low-HIV-prevalence comparator. These 11 groups of states consisted of composites of vast populations with considerable heterogeneity in sexual behaviors and circumcision prevalence (Table 1). Our primary research question involved assessing if HIV-related risk factors differed between high- and low-HIV-prevalence states. To avoid problems related to averaging out large differences in prevalence of risk factors in the groups of states, in our primary analysis we compared risk factors with estimated HIV prevalence by state. In a sensitivity analysis this analysis was repeated using the 11 groups of states.\n\nP<0.05 P<0.005 *P<0.0005\n\nHigh HIV prevalence states. UNAIDS defines populations with a 15-49-year-old HIV prevalence of greater than 1% as a generalized HIV epidemic21. We therefore defined states with a 15-49-year-old HIV prevalence of above 1% as high-HIV-prevalence states.\n\nLow HIV prevalence comparator state. Uttar Pradesh was chosen as the low-HIV-prevalence comparator state for a number of reasons. The previous HIV-serolinked NHFS, as well as the current NFHS survey, found an adult HIV prevalence of less than 0.1% in Uttar Pradesh15. It also has the largest population of any state in India and as a result it had the largest sample size of all states in NFHS-4. In sensitivity analyses we repeated the analyses using all Indian states with a HIV prevalence below 0.2% as the low HIV prevalence comparator population.\n\nAll HIV-prevalences were calculated for 15–49-year-olds.\n\nEach of following predictor variables were calculated separately for men and women and were limited to those between the ages of 15–49 years for women and 15–54 years for men.\n\nCondom use at last sex: The percentage of respondents who reported using a condom at last sex, amongst those who have had sex in the past 12 months.\n\nMale circumcision: The percentage of men who reported being circumcised.\n\nHigh-risk sex (sex with a non-married, non-cohabiting partner): The percentage of respondents who reported sex with a non-marital, non-cohabiting partner in the past 12 months, amongst all respondents who reported sex in the past 12 months.\n\nMultiple partners in the past year: The percentage with two or more sexual partners in the past 12 months amongst all respondents who reported sex in the past 12 months.\n\nLifetime sex partners: The mean number of reported lifetime sex partners amongst all respondents who reported having had sex, excluding those with greater than 90 partners.\n\nThe reason for excluding those with 90 or more partners was that in a small number of states these individuals exerted a large effect on the mean number of lifetime partners. Our primary research question was whether or not there were population differences in lifetime number of partners for the majority of the population. The percent of the population with 90 partners or more was small in all states: men, median 0.17 (IQR 0-0.29); women, median 0.39 (IQR 0.2-0.89) (Table 1). As a result we found it more informative to calculate mean lifetime number of partners excluding those with more than 90 partners.\n\nTwo socioeconomic control variables were assessed:\n\nEducation attained: Percent of state respondents whose highest educational attainment is primary or no schooling.\n\nPoverty: Percentage of the respondents from this state that were calculated to fall in the poorest 20% (quintile) of the nationally derived wealth band. The wealth quintiles were derived from an asset index.\n\n\nStatistical analysis\n\nAll analyses are ecological in nature and conducted with HIV prevalence by state or group of states as the outcome variable. The analyses were conducted using STATA 13.0 (College Station, TX) and were all adjusted to account for the complex sampling strategies of the survey using the survey (SVY) command. The analyses were stratified by gender. The two-sample Wilcoxon rank-sum test was used to assess if there was a difference in the median number of lifetime partners between the high-HIV-prevalence states and Uttar Pradesh/low-HIV-prevalence states. Cohen's d was used to assess effect size. Histograms were used to depict the distribution of the number of lifetime sexual partners by gender and state. Chi-squared tests were used to assess differences in categorical variables. Pearson’s correlation was used to assess the relationship between state-level HIV prevalence and the prevalence of each risk factor.\n\n\nResults\n\nAn overview of the sample size, mean ages and other demographic characteristics of men and women by state are provided in Table 1. The average age of respondents in the regions ranged from 28.9 to 34.8 years in men (median 32.0 [IQR 31.1-33.0]) and 31.4 to 35.5 years in women (median 33.0 [IQR 31.4-35.5]).\n\nThe overall 2015 HIV prevalence in 15–49-year-olds was 0.25% in men and 0.23% in women. The median HIV prevalence by state was 0.13% (IQR 0.05-0.48%) and the prevalence in Uttar Pradesh was 0.06% (Table 1).\n\nThe prevalence of HIV exceeded 1% in 5 states. Three of these were in the North East (Manipur, 1.2%; Nagaland, 1.4%; and Mizoram, 1.6%) and two elsewhere (Andhra Pradesh, 1.2%; and Chandigarh, 1.1%).\n\nCircumcision. The prevalence of circumcision was low in all states, including Uttar Pradesh (19.1%), but was significantly lower in all the high-HIV-prevalence states (2.5-10.6%; all P<0.0005).\n\nCondoms at last sex. Men/women in Andhra Pradesh (1.9/0.6%), Manipur (7.7/3.3%), Mizoram (9.1/2.9%) and Nagaland (10.5/3.1) were less likely, but those from Chandigarh (25.5/38.0%) were more likely to report using a condom at last sex than those from Uttar Pradesh (13.5/13.1%; all P-values <0.05).\n\nHigh-risk sex. The prevalence of reported high-risk sex in men was 8.3% in Uttar Pradesh. A higher proportion of men in Mizoram (20%) and Nagaland (15%) but a lower proportion in Andhra Pradesh (2.8%) reported higher risk sex (all P<0.0005). Reported proportions in women were low and did not differ between states.\n\nMultiple partners in past year. Men in Mizoram were more likely to report multiple partners (3.8%) in the past 12 months than Uttar Pradesh (1.5%) (P<0.005).\n\nLifetime partners. Men/women in Mizoram (3.39/1.12) and Nagaland (1.74/1.15) reported a higher number of lifetime partners than Uttar Pradesh (0.97/1.07); women in Andhra Pradesh reported fewer partners (1.01) (all P<0.0005). The magnitude of the right shifting in the distribution of lifetime number of partners was large for men in Mizoram and Nagaland (Cohen's d = 0.68 and 0.43, respectively) and small elsewhere (Table 2; Figure 2).\n\n(A) men aged 15–54 years and (B) women aged 15–49 years. Data from five high HIV prevalence states and Uttar Pradesh. Distributions are truncated at 10/6 partners for men/women.\n\nTwo sensitivity analyses were performed. Firstly, repeating the analyses using all Indian states with HIV prevalences below 0.2% as the low-HIV-prevalence comparator population (instead of Uttar Pradesh) had little effect on the results (Table 3). Secondly, repeating the analyses using the 11 groups of states in place of the 36 states produced comparable results (Table 4). Low circumcision and condom-usage rates together with lifetime number of partners and high-risk sex (men only) remained significant risk factors in group 7 states (Mizoram, Manipur, and Nagaland) compared to the Uttar Pradesh group (Uttar Pradesh, Madhya Pradesh, Uttarakhand, and Rajasthan).\n\nP<0.05 P<0.005 P<0.0005\n\nP-values are for comparisons between group 11 (low HIV prevalence group) and groups 1 and 7 (high-HIV-prevalence groups).\n\nP<0.05 P<0.005 P<0.0005.\n\nThe prevalence of higher-risk sex in men (r=0.40; P=0.017) and mean number of lifetime partners in men (r=0.55; P=0.001) were positively associated with HIV prevalence (Table 5).\n\nPearson’s correlation coefficients for the association between HIV prevalence and various risk factors by region in India's 36 States in NFHS-4.\n\n P<0.05, ** P<0.005. † These two variables were calculated with the combined men and women's education/poverty data and hence are the same for men and women. NA, not applicable.\n\n\nDiscussion\n\nAs in other countries, the spread of HIV has been heterogenous in India. Whilst the absolute differences in HIV prevalence by state are not large, the relative differences are. A state-level HIV prevalence above 1% is four times the national average prevalence. These differences in HIV prevalence are also fairly stable based on both data from the 2005 NFHS survey15 and other data sources such as antenatal surveys from the 2000s until the present13,16. Previous studies have argued that the higher prevalence rates in certain states in Northeast India is predominantly due to patterns of IVDU13,16. We were unable to investigate the role of IVDU, but we found population-level differences in the prevalence of circumcision, condom usage and sexual behaviors, which could explain differential HIV spread.\n\nThe most consistent association we found was lower circumcision prevalence rates in the high HIV prevalence states. Circumcision rates were, however, as low or lower in a number of low HIV prevalence states as in the high HIV prevalence states. This, plus the low circumcision prevalence throughout India, with little variance in absolute prevalence, suggest that circumcision may not play a dominant role in differential HIV spread in India. Although differences in condom usage were statistically significant, the absolute differences were small and one high-HIV-prevalence state had higher condom utilization than the low-HIV-prevalence comparators.\n\nThe relationship between the sexual behavioral risk factors and HIV prevalence was not uniform. Whilst sexual behaviors were in general riskier in the North East states, this was not the case in Andhra Pradesh and Chandigarh, where a number of the behavioural risk factors were actually less prevalent than Uttar Pradesh. In a country as vast and diverse as India, it should not be too surprising to find different combinations of risk factors to be responsible for HIV spread in different regions. As noted above, previous studies have suggested that various factors in Andhra Pradesh and Karnataka may play a role in the local HIV epidemics here13,20. An important finding of our study was the higher number of lifetime partners, multiple partners in the prior year and high-risk sex in Mizoram and Nagaland compared to Uttar Pradesh. Our study is thus compatible with the thesis that these behaviors would translate into a more connected sexual network which could play a role in the generation of higher HIV prevalence rates in these states.\n\nThese same three risk factors (number of lifetime partners, multiple partners in the prior year and high-risk sex) have previously been found to be associated with differences in HIV prevalence by ethnic group in Kenya3, South Africa5 and elsewhere8,22. Previous reports from DHS data found that, at an individual level, there was a stepwise increase in HIV prevalence with increasing lifetime sex partners in all 15 countries with available data. This was true for both men and women in all cases4,23. This association was also present in the Indian NFHS-3 in 2005 and in this survey14,15. That this association was present at both individual and population levels increases the likelihood that the association between lifetime sex partners and HIV prevalence is real24,25. In our study, this association was, however, only statistically significant for men. High-risk sex has also been found to be an individual level risk factor for HIV infection in a number of a number of countries, including India4,14. As in other areas, it is likely that these patterns of higher risk sexual behavior interact with other risk factors such as IVDU to produce the observed differences in HIV prevalence16,19.\n\nThis study has a number of limitations. The study is ecological in nature and is thus susceptible to the ecological inference fallacy. DHS surveys are not optimal for collecting sensitive sexual information26,27. The use of computer-assisted interviewing would be expected to have reduced but not eliminated this problem26. In addition, although the response rates for participation in the survey and HIV testing were high, this varied somewhat between states. The survey was designed to provide HIV prevalence estimates for 11 groups of states and not individual states. The data is thus susceptible to a large number of biases such as misclassification, nonresponse, recall and social desirability biases. In particular, other work has found evidence of culture-specific heterogeneity in answering questionnaires28,29. We cannot exclude the possibility that respondents from states where lower-risk sexual behavior was reported were subject to a greater social desirability bias, which could invalidate our findings. The available evidence, however, suggests that only minor differences in sexual behavior exist between those who do and do not answer sexual behavior questionnaires30. Finally, the analyses do not control for the influence of other variables.\n\n\nConclusion\n\nWe found a range of risk factors to be more prevalent in high-HIV-prevalence states in India. There was no clear single risk factor (or combination thereof) which appeared capable of explaining the heterogeneity of HIV spread in India. In the case of Mizoram and Nagaland, however, a higher prevalence of sexual risk behaviors may be contributing to higher HIV prevalence rates in this region. More detailed comparative studies between these populations and lower prevalence populations elsewhere in India may confirm or refute this finding.\n\nStudies of other higher-HIV-prevalence-populations that have managed to reduce HIV incidence, including Uganda, Zimbabwe, Kenya and Thailand, have pointed to the importance of reductions in multiple partnering in effecting this decline31–34. If other studies confirm our findings, then similar campaigns could be considered in Mizoram and Nagaland.\n\n\nData availability\n\nThe NFHS-4 survey is freely available from www.measureDHS.com as part of the India: Standard DHS, 2015–16. Access to the dataset requires registration, and is granted to those that wish to use the data for legitimate research purposes. A guide for how to apply for dataset access is available at: https://dhsprogram.com/data/Access-Instructions.cfm.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank Measure DHS for generously sharing the data.\n\n\nReferences\n\nAuvert B, Buvé A, Ferry B, et al.: Ecological and individual level analysis of risk factors for HIV infection in four urban populations in sub-Saharan Africa with different levels of HIV infection. AIDS. 2001; 15(Suppl 4): S15–30. PubMed Abstract\n\nGewirtzman A, Bobrick L, Conner K, et al.: Epidemiology of Sexually Transmitted Infections. In: Sexually transmitted infections and sexually transmitted diseases. edn. Edited by Gross G, Tyring SK. Heidelbergh: Springer Verlag, 2011; 13–35. 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Reference Source" }	[ { "id": "45246", "date": "12 Mar 2019", "name": "Curtis Jolly", "expertise": [ "Reviewer Expertise Immunology and Infectious Diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is simple and is a good start for a more complex study with more in-depth analyses. The author should describe the ecological zones in more detail. What makes zones homogenous or heterogeneous (poverty, sexual relations, business activities, etc.)? Can you relate ecological zones with the geographical, economic, ethnic etc., situation in India? The author reports that States were classified as low and high risks previously; it would be important to show a correlation between his/her selected States and the previous high or low risk States. The author states that the study was controlling for two socioeconomic variables (education, and poverty), but never indicated in the results and discussions how the controls affected the results. Simple correlation analyses were conducted, but the author mentions “likelihood” in stating that there is a greater likelihood of having high HIV prevalence if men were uncircumcised or had multiple partners. Greater or lower likelihood cannot be used with simple correlation. We can only talk of a relationship, to report likelihood the author would have to do further statistical analyses such as logi/probit/tobit models, and chance ratio. The results in Tables 3 should be divided between high and lower States. What are the high and low risk States? The signs of the coefficients should be mentioned, what does a positive sign signify? Each table should be fully understandable on its own.\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4696", "date": "17 Jun 2019", "name": "Chris Kenyon", "role": "Author Response", "response": "Thank you for this comment. We have provided more background information as the heterogeneity of peoples in India’s states (Page 3):India is a federal union comprising 29 states and 7 union territories (Figure 1). We refer to these 36 entities as states. The states are typically large and vary in surface area from Rajastan’s 342,239km2 to Goa’s 3,702km2 (median 88,752km2). The Union Territories are far smaller (median size 491km2) and for this reason are indicated with circles on the map in Figure 1. India’s vast size incorporates a wide range of ethnic and liguistic groups. It has an estimated 2000 ethnic groups and classifies 23 languages as official15. There are considerable differences between states in ethnic composition as well as in educational attainment, life expectancy and poverty rates14,15. This information has been added:India’s prior HIV prevalence survey (NFHS-3) in 2005-2006 was designed to provide HIV prevalence estimates from six states15. Of these, Manipur had the highest HIV prevalence (1.13%), followed by Andra Pradesh (0.97%), Karnataka (0.69%), Maharashtra (0.62%), Tamil Nadu (0.34%) and Uttar Pradesh (0.07%) in 15-49 year old men and women. This has been made clear in the new version which now reads (Page 8):Controlling for education and poverty levels, the prevalence of higher-risk sex in men (r=0.40; P=0.017) and mean number of lifetime partners in men (r=0.55; P=0.001) were positively associated with HIV prevalence (Table 4). The word ‘likelihood’ has been changed to ‘probability.’ Thank you for pointing this out. The five states that are classified as high HIV prevalence are now clearly listed in the title of Table 3. The list of all low HIV prevalence states would be too long to merit inclusion in the title, but the definition of low HIV prevalence state (HIV prevalence ≤0.2%) has been included in the title.Table 3. Comparison of prevalence of HIV-related risk factors in high HIV prevalence states (>1%; (Andhra Pradesh, Chandigarh, (Manipur, Mizoram and Nagaland) with low HIV prevalence states (≤0.2%).The Table contains prevalence figures of HIV-related risk factors and no coefficients. This has been made clear in the title and in the column headings." } ] }, { "id": "48166", "date": "10 Jun 2019", "name": "John McBride", "expertise": [ "Reviewer Expertise I am an infectious diseases physician with interests in the diagnosis of infectious diseases and various aspects of HIV including the role circumcision in the prevention of HIV." ], "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 provides some valuable insights into the transmission of HIV in India. The findings are consistent with other international studies in showing that an increased number of lifetime sexual partners and lower condom use is associated with higher HIV prevalence. It also confirms other ecological studies showing an inverse relationship between circumcision rate and HIV prevalence.\nThe data for the study was obtained from the NFHS-4 survey and in the methods section, where you discuss ethics, could you clarify that it is the survey that had ethical approval rather than this study.\nI did have some difficulty following the geography outlined in the paper. In Figure 1, some of the circles for union territories appear not to be associated with any particular area (e.g. DN). There is double labelling for DD and PY and LD is not in the legend. In the text there was mention of 11 groups of states which should have appeared in Table 2 but there are only five states mentioned in this table. Table 4 might be the more appropriate table. Table 2 and Table 3 are almost identical with only the last row different - you could probably get rid of one table by combining them.\nIn the discussion, whilst I accept that there is overlap of the circumcision rates between various states with high and low HIV prevalence I don't think there is enough data available in the study to support the contention that circumcision is not an important factor. I think the study supports high-risk sex and condom use as being the most important factors but in states where circumcision rates are low and HIV prevalence high - there should be some consideration of the role of circumcision in the public health response to HIV.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4695", "date": "17 Jun 2019", "name": "Chris Kenyon", "role": "Author Response", "response": "The wording as to the ethical approval process has been amended as suggested. The following text has been added to the methods section to assist the reader with understanding the difference in size between Union Territories and States:The states are typically large and vary in surface area from Rajastan’s 342,239km2 to Goa’s 3,702km2 (median 88,752km2). The Union Territories are far smaller (median size 491km2) and for this reason are indicated with circles on the map in Figure 1.As a result DN is indicated with a circle with no surrounding coloured area.The code for ‘LD’ is Lakshadweep and has been added. The double labelling for DD and PY has been rectified. This is a fair comment. To present a more balanced discussion of the role of circumcision, the discussion has been limited to the following:The most consistent association we found was lower circumcision prevalence rates in the high HIV prevalence states. Circumcision rates were, however, as low or lower in a number of low HIV prevalence states as in the high HIV prevalence states." } ] } ]	1	https://f1000research.com/articles/8-60
https://f1000research.com/articles/8-861/v1	13 Jun 19	{ "type": "Study Protocol", "title": "Cost-effectiveness of invasive devices versus non-invasive devices for screening of anemia in field settings in India: A study protocol", "authors": [ "Sutapa Bandyopadhyay Neogi", "Denny John", "Jyoti Sharma", "Rakhee Kar", "Sitanshu Sekhar Kar", "Maitreyee Bhattacharya", "Kartavya Tiwari", "Renu Saxena", "Denny John", "Jyoti Sharma", "Rakhee Kar", "Sitanshu Sekhar Kar", "Maitreyee Bhattacharya", "Kartavya Tiwari", "Renu Saxena" ], "abstract": "In India, an estimated 53% of women and 58% of children are anemic. The accuracy of Sahli’s hemoglobinometer, commonly used for detecting anemia in public health settings, is questionable. This study presents the protocol for assessment of cost and cost effectiveness of devices for screening of anemia using invasive devices (HemoCue 301 and True Hb), and non-invasive devices (AJO Spectroscopic Test and Masimo Pulse Oximetery test) compared to automated auto-analyser (reference test). The study population will include all adult patients attending the outpatient department in urban/rural health centres for routine investigations. Each included patient will undergo either one or two index tests apart from the reference test, on a predefined weekly schedule to avoid bias. The total and incremental costs of the intervention will be measured prospectively by measuring both screening and provider costs. Since the priority of the national program is detection of severe anemia, detection rates of anemia and severe anemia will be considered to calculate effectiveness. Cost comparisons of median, average and range of costs across the invasive and non-invasive devices will be calculated. Cost-effectiveness analysis will be compared for four devices within time horizon of 1 year. Ethics approval for the study has been obtained from the institutional ethics committees of the hospitals. The study protocol will generate evidence on the use of cost effectiveness of medical devices to influence policy decisions.", "keywords": [ "anemia", "invasive", "non-invasive", "costs", "cost-effectiveness", "India" ], "content": "Background\n\nAnemia is a public health problem that affects women and children, especially in low- and middle-income countries (LMICs). It affects nearly 53% of women and 58% of children in India1. The most commonly used parameter to diagnose and detect anemia is by measuring hemoglobin (Hb). Several methods have been tested to ascertain an accurate value of Hb that can guide the course of management.\n\nIn India, Sahli’s hemoglobinometer has been in use extensively in public health settings. Its accuracy is being questioned, and hence there is a felt need to replace this device by a more accurate and easier-to-use method. Currently, several invasive and non-invasive devices are available for detecting anemia. These have been evaluated in diverse settings and population groups with mixed findings2–4. In the absence of a concrete evidence, there is a need to examine the devices that have the potential to be included in public health programs. Given the fact that most of the screening happens in community and outreach settings where provision for a laboratory support seems difficult, the device ought to be tested in field settings with health workers (Auxillary Nurse Midwives or ANMs) as end users.\n\nCurrently four such devices have been identified that have the potential to be used in public health settings, namely, digital hemoglobinometers (True Hb and Hemocue), and non-invasive devices, i.e. Masimo Pulse Oximetry test and AJO spectroscopic device test5. There is a need to evaluate them in order to identify the most cost-effective device suitable for use in Indian and similar settings.\n\nFor the national Anemia Mukt Bharat (translated as ‘Anemia Free India’) program, diagnosis of anemia remains the mainstay for adequate and appropriate management. Public health programs require devices that are accurate, user friendly, and cost effective. A systematic approach using the principles of Health Technology Assessment (HTA) is required to identify the device that meets the requirements of health system6. A multi-disciplinary approach encompassing analytical frameworks, economic assessments and outcomes research forms the basis of HTA7. Until recently, few studies on HTA of medical devices have been conducted in LMICs8,9. In India, use of HTA for priority setting and decision making has been adopted recently. The current study protocol aims to compare costs, and cost-effectiveness of different hemoglobinometers in field settings.\n\nThe aim of the study is to compare the costs and cost-effectiveness of invasive (TrueHb and Hemocue) and non-invasive devices (Masimo and AJO Spectroscopic) to detect anemia in normal adults in field settings.\n\nPrimary objective. To estimate the cost-effectiveness of devices by evaluating the incremental cost per case detected from a health system perspective over a year’s time horizon based on the detection rate of anemia cases examined by invasive and non-invasive medical device methods for anemia in India.\n\nSecondary objectives.\n\na) To measure the health-related quality of life (HRQoL) using EQ-5D tool across invasive and non-invasive devices for testing anemia.\n\nb) To assess the user friendliness of the device for detection of anemia in field settings.\n\n\nMethods\n\nThis is a prospective diagnostic accuracy study conducted across two tertiary care hospitals India- Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, and Calcutta Medical College, Kolkata, West Bengal.\n\nThe study will be conducted in field settings of two sites in India in 2019. These sites will be Medical Colleges with a facility for performing the gold standard test and having a field practice area. The sites will be considered depending on the willingness of the Head of the Institution/ Department of Community Medicine.\n\nReference test (Gold standard). A hematological auto-analyzer will be selected as the reference test. These pieces of equipment are kept in Medical Colleges and are subjected to quality assessments through an internal and external quality checks for best results. Venous samples will be used for the tests.\n\nIndex tests. For invasive tests, Hb will be tested using both capillary and venous blood. The devices to be tested include HemoCue 301 and True Hb version. Non-invasive devices include AJO Spectroscopic Test and Masimo Pulse Oximetry Test. The details about these devices are described elsewhere5. Standardized methods will guide the performance of tests using these methods.\n\n\nStudy participants\n\nThe study population will include consecutive adult patients attending the outpatient departments (OPD) of urban and rural field practice areas attached to the select Medical Colleges. Children, pregnant women and patients suffering from bleeding disorders will be excluded from the study. All the patients will be recruited face to face at the clinics. Only those patients who are advised Hb estimation as part of the routine investigations will be screened for their eligibility for the study. They will undergo the reference (Gold standard) test. In addition, each patient will be subjected to one or two index tests. Allocation of participants to different index tests will be based on a predefined weekly schedule5.\n\nA sample of size of 600 is required to assess the diagnostic accuracy of each device based on a 50% prevalence of anemia, sensitivity = 82% (TrueHb) and 82% (HemoCue, at 5% level of significance. Details of the calculation have been mentioned elsewhere5.\n\n\nData collection\n\nIndividual level data on Hb readings of autoanalyzer and other devices will be collected electronically. Data from field workers will be collected on paper forms. Data pertaining to every participant will be linked using a unique identification number given at the time of recruitment. Blinding will be ensured by allowing users to upload the data real time that will be merged at the central level using unique identification number. This will help maintain the validity of the study5. Missing data or indeterminant data of the index test or reference standards will be excluded from the analysis.\n\n\nCost assessment\n\nWe will capture the costs using a step-down costing methodology on paper forms, that will be transferred to a customized tool created in MS Excel, available as Extended data10.\n\nUnit costs of all resources used in the different activities occurring in the in OPDs for the purpose of screening will be collected. The cost of ANM time will be calculated based on the time they would devote for testing for anemia including noting down patient details and test results. The costs will include full salary including fringe benefits.\n\nThe life-cycle of each medical device, considered as capital equipment in the study, will be 2.5 years. Annual maintenance cost of all diagnostic equipment will be assumed to be 10% of the annual rental value. Costs of other equipment such as syringe, needle, cotton swab and other consumables for conducting each test will also be accounted for.\n\nThe rental value of clinics (urban and rural health centers) will be captured based on present value as per the local information. Contribution of rental values of clinics and capital equipment towards each test will be calculated based on the time required to perform the test11.\n\nTo determine the examination cost, we will prepare the list of equipment needed to conduct each test. At the hospital, the examination cost will include both direct and overhead costs. Direct costs will include labor, capital and material costs. Labor cost will be the cost of ANM time, calculated based on the time they would devote for testing for anemia including noting down patient details and test results. Average time taken by ANMs to perform the test based on real-time observations of at least 30 patients would be captured. The costs will include full salary including fringe benefits. Capital costs will include annualised discounted depreciation cost of furniture, equipment and instruments used in the OPDs for screening. The material costs will include the actual usage of drugs, medical supplies, and office supplies for performing each test. Any wastage of resources incurred in the process will be accounted for. These costs to the public health system will be elicited from the records/ publications.\n\nOverhead costs will not be considered as both the facilities being large public hospitals, are managed through government funding; it will be difficult to identify budgetary allocations, and expenditure heads for allocation towards examination cost for each test.\n\nThe cost data will be collected in a MS-Excel divided into eight sections named as under:\n\ni. Information about the facility: basic details of the facility will be collected.\n\nii. General information: which include data about working hours of the health facility\n\niii. Salary structure of the ANM as reported\n\niv. Details of Equipment: The cost of devices will be directly obtained from the manufacturers in the prescribed format. The format will include name of the device, unit price and expected life of the equipment. This data will be collected across the sites in Kolkata and Puducherry. The prices provided by the manufacturer will be considered for cost of screening.\n\nv. Consumables: Under this section, details of consumables which includes materials and supplies issued, consumed, quantity used per test, and price per unit for every device will be collected in the facility.\n\nvi. Details of the physical infrastructure: Under this section area, monthly rental price of 100 square feet place where the center is located and expenditure (if any) on renovation or construction of accessory items will be collected.\n\nvii. Details about non-medical items: Name and quantity of functional non-medical items in each room will be physically observed by the researcher and reported.\n\nviii. Activity time: Under this the respondents will be asked to report the activities which they perform and the time they spend routinely for the same. At both the sites the time which the ANM take for conducting the test will be estimated.\n\nThe costs will be presented as average values across each medical device. The proportion of cost distribution across human resources (ANM), equipment (device, charger and adapter), accessories (microcuvettes/strips), consumables (items used in the test), non-medical (items in examination room) and examination room (rental value) will also be presented.\n\n\nMeasurement of cost-effectiveness\n\nCost-effectiveness analysis will assess the relative efficiency of the invasive and non-invasive methods for detecting anemia. Standard protocols for conducting such evaluation of diagnostic tests would be used12. This will be reported as ‘correct diagnosis’ as the outcome measure. Correct diagnosis would be defined as ‘agreement of the result of the particular diagnostic method with the gold standard’ or detection rate5. The detection rate will be the sensitivity of the devices i.e. ability of the devices to identify true positives. Since the priority of the national program is detection of severe anemia, we will consider the detection rates of anemia and severe anemia to calculate effectiveness.\n\nThe health-related quality of life (HRQoL) of patients will be measured at the time of enrolment into the study using the EQ- 5D-5L questionnaire13.\n\nAdditionally, the user friendliness across key attributes (ease of use, efficiency in daylight, scope of subjective errors, portability, convenience to patient, interpretation of Hb results, need for power/battery, average time taken for performing one test, and expertise required) will also be analyzed.\n\nGeneral principles of cost-effectiveness will be applied to the results of costs and detection rate of each device14. The device with best accuracy results (sensitivity in this case) will be taken as the reference. Then it will be determined if this device was dominated by other devices. A dominated device is more costly, but less effective than another device. For non-dominated devices, the cost-effectiveness analysis will combine the unit costs per detection rate, i.e. the incremental cost effectiveness ratio (ICER).\n\nThe ICER will be calculated as: [(Mean cost per test) device A − (Mean cost per test) device B] / [(Detection rate) device A − (Detection rate) device B].\n\nICER values will be presented using costs and detection rates of each device, and separately for detection of anemia and severe anemia.\n\nThe results of cost-effectiveness analysis would be presented as incremental cost per detection rate of one method compared to the other. Results would be presented in a manner enabling easy comparison between all diagnostic techniques to assess relative efficiency.\n\n\nLong-term benefits of anemia screening\n\nThe long-term benefits and cost-effectiveness of early detection of anemia comparing invasive and non-invasive methods will be analysed using decision analytical model, by quantifying the contribution of screening in preventing morbidities using data from Indian studies.\n\n\nAssessment of health-related quality of life (HRQoL)\n\nHRQoL will be assessed using a standardized method. Five domains, namely mobility, self-care, usual activities, pain/discomfort, anxiety/depression will be considered. Each one will be given a score ranging from 1 to 5, with 1 being the worst and 5 the best. The response of five questions of EQ-5D-5L questionnaire will be converted into a single index value as described in the methodology of the questionnaire13.\n\n\nUser friendliness\n\nLaboratory technicians and ANMs will independently rate each of the following criteria: ease of use, efficiency in daylight, scope of subjective errors, portability, convenience to patient, interpretation of Hb results, need for power/battery, average time taken for performing one test, and expertise required, on a scale of 1 (very poor) to 5 (excellent) for each device method. The total score across each of these parameters for laboratory technicians and ANMs will be used to identify user friendliness for each method (maximum score for each method.\n\n\nSensitivity analysis\n\nOne-way and probabilistic sensitivity analysis will be conducted using lower and upper bound of sensitivity values of each device, and other cost parameters (e.g. accessories, consumables etc.). Sequential tornado analysis will be presented using ICER values from the sensitivity analysis in order to rank-order the different variables that influence the cost per detection rate values15. Scenario analysis will be conducted for detailing the annual costs conducted by 1 ANM in a health facility for detecting anemia and severe anemia.\n\nReporting of results will be based on Consolidated Health Economic Evaluation Reporting Standards (CHEERS) guidelines for reporting health economic evaluation studies16.\n\nAnnexure 2, available as Extended data, provides details the parameters of the proposed data analysis10.\n\n\nEthical approval\n\nData collection will be started after obtaining ethics clearance from the Institutional Ethics Committee of the Indian Institute of Public Health, Delhi (IIPHD), All India Institute of Medical sciences (AIIMS), New Delhi and two recruiting sites (JIPMER, Calcutta Medical College). Written informed consent will be obtained from every eligible participant. Information collected will not have access to anyone else other than the research team. The treatment of patients will be based on the readings of the reference test and will in no way influence the results of the index test.\n\n\nConclusion\n\nThis paper constitutes a protocol for the costs and cost effectiveness of medical devices in a LMIC. The evidence generated will contribute significantly by lending evidence on the use of cost effectiveness to influence decisions.\n\n\nData availability\n\nNo underlying data are associated with this study.\n\nOpen Science Format: Cost-effectiveness of invasive devices versus non-invasive devices for screening of anemia in field settings in India: A study protocol. https://doi.org/10.17605/OSF.IO/DJUV510.\n\nThis project contains the following extended data:\n\nAnnexure 1.xlsx (step-down costing methodology).\n\nAnnexure 2.xlsx (parameters proposed to be used in data analysis).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe study is being funded by Department of Health Research (Health Technology Assessment division, Sanction number F.NO.S.11011/02/2017- HR), Ministry of Health and Family Welfare, Government of India.\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\nIIPS: National Family Health Survey 4. Mumbai, India: 2015-16. Reference Source\n\nHinnouho GM, Barffour MA, Wessells KR, et al.: Comparison of haemoglobin assessments by HemoCue and two automated haematology analysers in young Laotian children. J Clin Pathol. 2018; 71(6): 532–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahajan PB, Mukherjee S: Role of point of care Hb diagnostic devices in getting the right picture of anemia control: Tangi Rural Anemia Diagnostic Accuracy Study. J Drug Assess. 2018; 7(1): 34–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParker M, Han Z, Abu-Haydar E, et al.: An evaluation of hemoglobin measurement tools and their accuracy and reliability when screening for child anemia in Rwanda: A randomized study. PLoS One. 2018; 13(1): e0187663. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeogi S, Negandhi H, Sharma J, et al.: Diagnostic efficacy of digital hemoglobinometer (TrueHb), HemoCue and non invasive devices for screening patients for anemia in the field settings-a proposal. Indian J Comm Health. 2018; 30(Supp): 86–8. Reference Source\n\nWHO: Health technology assessment of medical devices. Geneva: 2011. Reference Source\n\nFacey K: Health Technology Assessment (HTA) Glossary. In: Assessment INoAfHT, editor. Sweden, 2006. Reference Source\n\nMcLaren ZM, Sharp A, Hessburg JP, et al.: Cost effectiveness of medical devices to diagnose pre-eclampsia in low-resource settings. Dev Eng. 2017; 2: 99–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTyrrell A, Worrall E, Que TN, et al.: Cost and effectiveness comparison of two methods for screening potential blood donors for anaemia in Vietnam. Transfus Med. 2011; 21(3): 158–65. PubMed Abstract \| Publisher Full Text\n\nNeogi SB: Cost-effectiveness of invasive devices versus non-invasive devices for screening of anemia in field settings in India: A study protocol. 2019. http://www.doi.org/10.17605/OSF.IO/DJUV5\n\nJohn D, Parikh R: Cost-effectiveness and cost utility of community screening for glaucoma in urban India. Public Health. 2017; 148: 37–48. PubMed Abstract \| Publisher Full Text\n\nSanghera S, Orlando R, Roberts T: Economic evaluations and diagnostic testing: an illustrative case study approach. Int J Technol Assess Health Care. 2013; 29(1): 53–60. PubMed Abstract \| Publisher Full Text\n\nEQ-5D-5L User Guide: basic information on how to use the EQ-5D-5L instrument. editor, 2015; [cited 2019]. Reference Source\n\nDrummond MF, Sculpher MJ, Torrance GW, et al.: Methods for the Economic Evaluation of Health Care Programmes. Oxford University Press; 2005. Reference Source\n\nMuenning P: Cost effectiveness analysis in health: A practical approach. 2nd ed. San Francisco, CA: Jossey-Bass; 2008. Reference Source\n\nHusereau D, Drummond M, Petrou S, et al.: Consolidated Health Economic Evaluation Reporting Standards (CHEERS)--explanation and elaboration: a report of the ISPOR Health Economic Evaluation Publication Guidelines Good Reporting Practices Task Force. Value Health. 2013; 16(2): 231–50. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49867", "date": "20 Jun 2019", "name": "Anil Gumber", "expertise": [ "Reviewer Expertise Economic Evaluation of health intervention/service/program" ], "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 protocol is written well. However, authors need to mention clearly how this protocol is different from the one published in 2018. This has been referred as: Neogi S, Negandhi H, Sharma J,\net al.: Diagnostic efficacy of digital hemoglobinometer (TrueHb), HemoCue and non invasive devices for screening patients for anemia in the field settings-a proposal. Indian J Comm Health. 2018; 30(Supp): 86–81. Beside comparing three devices in previously published protocol, what other generic features are included in this protocol. Currently, to me, its just an updated version of previously published protocol and thus should not be indexed again. Instead authors should have mentioned what progress they have made since publishing their previous protocol and how many individuals have been screened until now.\nIn the methods section, canvassing of EQ-5D questionnaire is mentioned but in cost-effectiveness analysis this information has not been used. It is not clear to me why this instrument is required (to measure changes in HRQoL or QALYs between individuals screened by invasive and non-invasive methods, which doesn't make any sense to me when individuals who are screened are not going to be followed-up).\nMore importantly, the very generic concept of Incremental Cost-Effectiveness Ratio (ICER) can't be applied here due to absence of follow-ups (i.e. no data is collected at two points in time). Authors can only compare cost per correctly detected screening outcome between four types of method/equipment instead of computing ICER.\nAuthors have not included details of costing information for enhanced training to field workers/ANMs to be used for various screening equipment in the clinical/community setting.\nFinally, there is a huge difference in purchase prices between equipment and how these will be used in cost-effectiveness analysis is not clear to me. For instance within invasive method, the cost of Tru Hb equipment is 8-10 times higher than HemoCue. And if one is just comparing the purchase cost per correctly detected screening outcome within invasive method would be a terrible blunder. Therefore, one needs to account for the case loads as well as the mixed-use for varied purposes for an equipment during its lifetime or becoming obsolete.\n\nI think these are serious issues in the published protocol and most things are replicated from the previously published protocol.\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": "153386", "date": "16 Nov 2022", "name": "Ifeoma P. Ijei", "expertise": [ "Reviewer Expertise Haemoglobin disorders", "laboratory quality management systems" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors reiterate the public health significance of anaemia in LMICs and the complexities of the modalities available for haemoglobin measurement in various clinical settings, from the capital-intensive, technically demanding haematology analyzers to hand-held, point-of-care testing devices. The cost-effective determination of anaemia remains pertinent and relevant especially in LMIC settings with high prevalence of anaemia and this, they have demonstrated in their introductory comments.\nThe authors have sought to compare the cost effectiveness of available technology for screening of anaemia in the field. The rationale aligns with clinical and policy needs for a quick, accurate, reliable and cost-effective method for the detection of anaemia which is highly prevalent in the study population and this is borne out in the primary objective of this study.\nThe measurement of HRQoL using EQ-5D tool however, may be reflective of the underlying aetiology of the anaemia detected rather than the nature of the technology (invasive and/or non-invasive devices) utilized in the detection of anaemia. Its determination as a secondary objective may not be relevant to this particular study.\nThe strategy for analysis of cost does not fall under the reviewer’s area of expertise but their exploration of the factors contributing to the evaluation of cost-effectiveness and factors impacting on end-user utilization is quite comprehensive.\nHowever, the opening statement for the study design denotes an evaluation of diagnostic accuracy of testing methodology which is not reflective of the title and main text. It is also unclear if the authors wish to demonstrate the cost-effectiveness or otherwise of the gold standard/reference test (auto-analyzer) against the index invasive and non-invasive test techniques or if the latter are being compared to each other.\nAdequate steps have been taken to satisfactorily address ethical concerns. The authors should indicate the potential levels of policy impact of this manuscript as this could be a significant milestone deliverable from this study.\nThe article is well written and makes for an interesting read. The authors would be well served by providing clarity to the aforementioned observations.\nReviewer Guidelines: Title: Is it reflective of the objective and design of the study?\n\nYes, the title is reflective of the objective and design of the study. Are the keywords searchable? Yes, the keywords are searchable.\n\nAbstract: Are the contents a comprehensive representation of the full text in terms of methodology, findings and conclusion? Is the volume satisfactory? The content of the abstract represents the text and is satisfactorily voluminous.\nIntroduction: Is the literature rich with global, regional and local literatures and perspectives? The literature provided predominantly reflects a local perspective. Is the gap in knowledge that the study is attempting to close obvious? Yes.\nMethods: Are ethical issues (consent, concealment of subject identity, institutional ethical clearance) well addressed? Yes, these are satisfactorily addressed. Is the study design consistent with the stated objective? Partly.\nResults: Is there internal consistency – do figures add up? Are there unexplained missing data? Are the Tables and figures simple and clear to understand? This information is not available for review.\n\nDiscussion: Are differences or similarities between comparison studies well explained? Are the issues discussed consistent with the findings in the study? This information is not available for review.\nConclusion: Are the conclusions based on the findings in the study? Are the recommendations based strictly on the findings in the study? This information is not available for review.\nReferences: Are the references adequate and satisfactorily current? Yes.\nGeneral: Is the entire text satisfactory in terms of spellings, use of punctuation, grammar and style of expression? Yes. Does the manuscript make a substantial contribution to knowledge? Yes, the manuscript has the potential to impact policy direction with some revision.\n\nVerdict: Accept with major revisions.\nGuidelines for making a Verdict: Acceptance with major revisions - Need for thorough copy-editing for spellings and grammar, data re-analysis, need for more tables or graphical expressions, need for more references or reduction of references, more discussion points, extensive reduction or expansion of the text.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] } ]	1	https://f1000research.com/articles/8-861
https://f1000research.com/articles/8-857/v1	13 Jun 19	{ "type": "Case Report", "title": "Case Report: A case of dipylidiasis in a first-trimester pregnant woman attending a routine antenatal clinic at Elmina health centre, Ghana", "authors": [ "Kwame Kumi Asare", "Yeboah Kwaku Opoku", "Alberta Serwah Anning", "Justice Afrifa", "Eric Ofori Gyamerah", "Yeboah Kwaku Opoku", "Alberta Serwah Anning", "Justice Afrifa", "Eric Ofori Gyamerah" ], "abstract": "Dipylidiasis is a zoonotic parasitosis caused by a canine and feline tapeworm, Dipylidium caninum which rarely infects humans, usually infants and young children. The accidental ingestion of cysticercoid infected flea Ctenocephalides felis is the cause of this cestode infection in humans. Here we report the first and a rare case of adult dipylidiasis in a first-trimester pregnant woman in Ghana. She reported at the health facility for a routine antenatal check-up with apparently no symptoms or signs of the infection at the time of the visit. Her routine stool examination revealed a single egg packet of Dipylidium caninum and was treated with a single dose of praziquantel. It is important for pathologists and laboratory technicians to be aware of the emergence of human dipylidiasis in Ghana. A conscious effort should be aimed at the creation of awareness among pet owners and the general population of the public health importance of zoonotic parasites that infect pets and domestic animals.", "keywords": [ "Human dipylidiasis", "canine & feline tapeworm", "pregnancy", "egg packets", "praziquantel", "Elmina", "Ghana" ], "content": "Abbreviations\n\nHB: Haemoglobin concentration; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular haemoglobin concentration; MCV: Mean corpuscular volume; WBC: White blood cell count.\n\n\nIntroduction\n\nHuman dipylidiasis is a rare zoonotic parasitosis caused by cosmopolitan dog tapeworm Dipylidium caninum (flea tapeworm or double-pore tapeworm)1. Previous reports of this accidental cestode infection had been from Asia, South American, Europe and North American countries2,3. At present, only 349 human cases of dipylidiasis have been reported worldwide after its first identification and description in 17584. Accidental infection of cysticercoid of Dipylidium caninum from ingestion of infected dog or cat fleas is the cause of this infection5. Most of the cases of this infection has been reported in children below five years likely kissed or licked by infected pets6,7. Rarely has an adult infection of dipylidiasis been reported in literature8,9.\n\nThe adult Dipylidium caninum of the order Cyclophyllidea and subclass Eucestoda produces gravid proglottids in the small intestine of dogs and cats which are the definitive hosts10. The proglottids rupture and expel packets of eggs after it has detached and migrated to the perianal region or is passed with faeces11. The larval stage of the intermediate host; Ctenocephalides felis, Ctenocephalides canis, Pulex irritans and Trichodectes canis ingest the hexacanth embryonic eggs of Dipylidium caninum which then develops into infective cysticercoid in the adult stage of the flea12. Cats and dogs which are definitive hosts or humans the accidental host become infected by ingesting a flea with cysticercoid13,14.\n\nHuman dipylidiasis is usually asymptomatic with occasional non-specific symptoms such as diarrhoea, nocturnal pruritus, anorexia, pruritus ani, urticaria, weight loss, epigastric pain, constipation and loss of appetite2,15. Heavily infected cats which are definitive host sometimes exhibit severe symptoms such as intestinal obstruction, epileptiform seizures and convulsion16,17. However, such severe forms of dipylidiasis have not been reported in human cases.\n\nA typical clinical diagnosis of Dipylidium caninum is by the identification of characteristic proglottids or egg packets and the cucumber seed-like body segments of the adult worm in stool18,19. However, egg packets rapidly disintegrated in the stools and can occasionally be found in fresh samples11,19. However, the uncommon nature, poor description by an immediate observer and little or no experience by laboratory professionals with Dipylidium caninum may render it undiagnosed20.\n\nAlthough human dipylidiasis has occasionally been reported in young children it has rarely been reported in adults, with exception of a 26-year-old kidney transplanted patient and a 57-year old woman8,9,21. Until now, there has not been any case of dipylidiasis associated with pregnancy reported in literature. In this article, we present the first case of dipylidiasis in a 27-year-old woman in her first trimester of pregnancy attending a routine antenatal care at Elmina healthcare centre, Ghana.\n\n\nCase presentation\n\nA 27-year-old first-trimester pregnant woman, a petty trader and an Akan from Komenda in Komenda-Edina-Eguafo-Abrem district in Central region of Ghana who visited the antenatal clinic in April 2013 for a routine antenatal check-up at Elimina healthcare centre in the Central Region of Ghana with no symptoms or signs of infection or abnormalities. She had experienced intermittent vomiting, slight headache and bloated stomach in the previous three weeks before visiting the antenatal clinic. Physical examination revealed she had a normal pregnancy with no clinical manifestation or any signs of threat to her health. A routine laboratory examination of full blood count (FBC), stool, urine, blood film for malaria parasites together with a serological test for syphilis and HIV were requested. She had no history of close contact with cats and dogs.\n\n\nLaboratory results\n\nHer laboratory test results were as follows (normal ranges are indicated in parentheses): HB, 10.2g/dL (10–14 g/dL); WBC, 5.3×109/L (2.2-8 ×109/L); MCV, 80.1fL (78–90 fL); MCH, 24.0pg (21–32 pg); MCHC, 31.7g/dL (30–34 g/dL); PLT, 247×109/L (150-450×109/L); neutrophils, 58.9% (40–60%); lymphocytes, 26.7% (20–40%); monocytes, 1.2% (2–8%); basophils, 0.8% (0.5–1%) and eosinophils of 12.4% (1–4%). Her urine, blood film analysis and the serological tests were all normal. Microscopic examination of direct stool samples revealed a single egg with packets of Dipylidium caninum which measured about 45 × 90 μm without any white blood cells in the stool sample. She was diagnosed with dipylidiasis and treated with a single dose of praziquantel orally (600 mg). The patient was followed until successful delivery without any complications.\n\n\nDiscussion\n\nThere are sporadic reports of human dipylidiasis in recent times, although humans are not the natural host22,23. The majority of these reports have been in infants and young children making them a high-risk group for Dipylidium caninum infection24–28. In this report, we present the first and rare case of dipylidiasis in a pregnant woman in her first trimester with no clinical symptoms at the time of antenatal examination in Elmina, Ghana. Despite the fact that the patient does not own a cat or dog as a pet, and had no history of contact with these animals which are the sources of accidental infection, she however stays in an area where dogs and cats stray freely without being confined by their owners.\n\nDipylidium caninum is a worldwide neglected helminth infection of dogs and cats, its natural hosts23,29. A study conducted in the Greater Accra region of Ghana reported a high prevalence of Dipylidium caninum as one of three zoonotic helminths of dogs that are kept for hunting and security purposes without confinement30. The free-range breeding of dogs and cats in Ghana without any control creates room for the contamination of the vicinity with their stool. These animals are usually not subjected to veterinary check-up and treatment; coupled with the fact that they feed in unhygienic places results in them posing a great environmental challenge further facilitating the transmission of infective fleas31. This rare case of dipylidiasis in pregnancy is a typical case of environmental transmission of Dipylidium caninum and unconscious ingestion of an infected flea. Despite the public health risk posed by Dipylidium caninum, many pet owners are ignorant to this parasitic infection30,32. Accurate diagnosis of dipylidiasis in humans is challenged by several factors which include the experience of the pathologist or laboratory technologist with Dipylidium caninum and the asymptomatic nature of the infection leading to many undiagnosed carriers. Further challenges arise due to the similar presentation of Dipylidium caninum with Enterobius vermicularis, and the lack of clear differential symptoms for Dipylidium caninum32,33. Clinical diagnosis is based on detection of proglottids or occasionally egg packets which usually rapidly disintegrate in stool34,35. Therefore, multiple stool examinations are sometimes required to detect proglottids or eggs in stool specimen35,36. Characteristically, Dipylidium caninum egg packets contain double genital pores which can be seen in unstained samples under a light microscope37. The adult worm possesses an ovigerous capsulated uterus with simple genitalia, armed rostellum with unarmed suckers and two-pore gravid proglottids6,37. These characteristics are important for differentiating Dipylidium caninum from other Cestodes such as Taenia solium, T. saginata and Hymenolepis nana38.\n\nCurrent anti-helminthic drugs such as praziquantel or niclosamide are highly efficient in the complete elimination of Dipylidium caninum in humans, dogs or cats37,38. Other treatment options for Dipylidium caninum include albendazole, mebendazole, thiabendazole, paromomycin, pyrantel pamoate and paromomycin19,39.\n\nA conscious effort should be aimed at the creation of awareness among pet owners and the general population of the public health importance of zoonotic parasites that infect pets and domestic animals.\n\n\nConclusion\n\nWe report the first and a rare human diphylidiasis in pregnancy from Ghana. To our understanding, this is the first case of human and adult infection of Diphylidium caninum from Ghana which makes it a case of public health concern. Therefore, the high prevalence of Dipylidium caninum infection among unconfined pets in communities inhabited by quite a number of young children in Ghana should be a public health concern.\n\n\nConsent\n\nA written consent was obtained from the patient for publication of this case report and its 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": "Grant information\n\nThis work was supported by the Department of Biomedical Science, College of Allied Health Sciences, University of Cape Coast.\n\n\nAcknowledgement\n\nWe are grateful to the laboratory and the medical staff of Elmina Urban Health Centre for their support.\n\n\nReferences\n\nMani I, Maguire JH: Small animal zoonoses and immuncompromised pet owners. Top Companion Anim Med. 2009; 24(4): 164–74. PubMed Abstract \| Publisher Full Text\n\nCraig P, Ito A: Intestinal cestodes. Curr Opin Infect Dis. 2007; 20(5): 524–32. PubMed Abstract \| Publisher Full Text\n\nMcCall JW, Genchi C, Kramer LH, et al.: Heartworm disease in animals and humans. Adv Parasitol. 2008; 66: 193–285. PubMed Abstract \| Publisher Full Text\n\nDantas-Torres F: Canine vector-borne diseases in Brazil. Parasit Vectors. 2008; 1(1): 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMolina CP, Ogburn J, Adegboyega P: Infection by Dipylidium caninum in an infant. Arch Pathol Lab Med. 2003; 127(3): e157–9. PubMed Abstract\n\nCabello RR, Ruiz AC, Feregrino RR, et al.: Dipylidium caninum infection. BMJ Case Rep. 2011; 2011: pii: bcr0720114510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurner JA: Human dipylidiasis (dog tapeworm infection) in the United States. J Pediatr. 1962; 61(5): 763–8. PubMed Abstract \| Publisher Full Text\n\nJackson D, Crozier WJ, Andersen SE, et al.: Dipylidiasis in a 57-year-old woman. Med J Aust. 1977; 2(22): 740–1. PubMed Abstract\n\nSahin I, Köz S, Atambay M, et al.: A Rare Cause of Diarrhea in a Kidney Transplant Recipient: Dipylidium caninum. Transplant Proc. Elsevier. 2015; 47(7): 2243–2244. PubMed Abstract \| Publisher Full Text\n\nMehlhorn H: Worms (Helminths). In Animal Parasites. Springer, Cham. 2016; 251–498. Publisher Full Text\n\nJiang P, Zhang X, Liu RD, et al.: A Human Case of Zoonotic Dog Tapeworm, Dipylidium caninum (Eucestoda: Dilepidiidae), in China. Korean J Parasitol. 2017; 55(1): 61–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConboy G: Cestodes of dogs and cats in North America. Vet Clin North Am Small Anim Pract. 2009; 39(6): 1075–90, vi. PubMed Abstract \| Publisher Full Text\n\nRobertson ID, Thompson RC: Enteric parasitic zoonoses of domesticated dogs and cats. Microbes Infect. 2002; 4(8): 867–73. PubMed Abstract \| Publisher Full Text\n\nGuzman RF: A survey of cats and dogs for fleas: with particular reference to their role as intermediate hosts of Dipylidium caninum. N Z Vet J. 1984; 32(5): 71–3. PubMed Abstract \| Publisher Full Text\n\nRabinowitz PM, Gordon Z, Odofin L: Pet-related infections. Am Fam Physician. 2007; 76(9): 1314–22. PubMed Abstract\n\nNappi AJ: Parasites of medical importance. CRC Press; 2002. Publisher Full Text\n\nMin DY: Cestode infections in Korea. Kisaengchunghak Chapchi. 1990; 28 Suppl: 123–44. PubMed Abstract \| Publisher Full Text\n\nSamkari A, Kiska DL, Riddell SW, et al.: Dipylidium caninum mimicking recurrent Enterobius vermicularis (pinworm) infection. Clin Pediatr (Phila). 2008; 47(4): 397–9. PubMed Abstract \| Publisher Full Text\n\nSzwaja B, Romański L, Zabczyk M: A case of Dipylidium caninum infection in a child from the southeastern Poland. Wiad Parazytol. 2011; 57(3): 175–8. PubMed Abstract\n\nHill P, Warman S, Shawcross G: 100 top consultations in small animal general practice. John Wiley & Sons; 2011. Reference Source\n\nRamana KV, Rao SD, Rao R, et al.: Human Dipylidiasis: A Case Report of Dipylidium caninum Infection from Karimnagar. Online J Health Allied Sci. 2011; 10(2). Reference Source\n\nGarcía-Agudo L, García-Martos P, Rodríguez-Iglesias M: Dipylidium caninum infection in an infant: a rare case report and literature review. Asian Pac J Trop Biomed. 2014; 4(Supplement 2): S565–7. Publisher Full Text\n\nMcCarthy J, Moore TA: Emerging helminth zoonoses. Int J Parasitol. 2000; 30(12–13): 1351–60. PubMed Abstract \| Publisher Full Text\n\nHotez PJ, Brindley PJ, Bethony JM, et al.: Helminth infections: the great neglected tropical diseases. J Clin Invest. 2008; 118(4): 1311–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBethony J, Brooker S, Albonico M, et al.: Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet. 2006; 367(9521): 1521–32. PubMed Abstract \| Publisher Full Text\n\nRobertson ID, Irwin PJ, Lymbery AJ, et al.: The role of companion animals in the emergence of parasitic zoonoses. Int J Parasitol. 2000; 30(12–13): 1369–77. PubMed Abstract \| Publisher Full Text\n\nDobler G, Pfeffer M: Fleas as parasites of the family Canidae. Parasit Vectors. 2011; 4(1): 139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNarasimham MV, Panda P, Mohanty I, et al.: Dipylidium caninum infection in a child: a rare case report. Indian J Med Microbiol. 2013; 31(1): 82–4. PubMed Abstract \| Publisher Full Text\n\nKlimpel S, Heukelbach J, Pothmann D, et al.: Gastrointestinal and ectoparasites from urban stray dogs in Fortaleza (Brazil): high infection risk for humans? Parasitol Res. 2010; 107(3): 713–9. PubMed Abstract \| Publisher Full Text\n\nJohnson SA, Gakuya DW, Mbuthia PG, et al.: Prevalence of gastrointestinal helminths and management practices for dogs in the Greater Accra region of Ghana. Heliyon. 2015; 1(1): e00023. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmissah-Reynolds PK, Monney I, Adowah LM, et al.: Prevalence of Helminths in Dogs and Owners' Awareness of Zoonotic Diseases in Mampong, Ashanti, Ghana. J Parasitol Res. 2016; 2016: 1715924. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathison BA, Pritt BS: A Systematic Overview of Zoonotic Helminth Infections in North America. Lab Med. 2018; 49(4): e61–e93. PubMed Abstract \| Publisher Full Text\n\nHurrell JM, Genta RM, Melton SD: Histopathologic diagnosis of eosinophilic conditions in the gastrointestinal tract. Adv Anat Pathol. 2011; 18(5): 335–48. PubMed Abstract \| Publisher Full Text\n\nWeese JS, Fulford M, editors: Companion animal zoonoses. John Wiley & Sons; 2010. Publisher Full Text\n\nKaufmann J: Parasitic infections of domestic animals: a diagnostic manual. Birkhäuser; 2013. Publisher Full Text\n\nWeisse ME, Mullins JK, Moffett KS: A neonate with worms. Clin Infect Dis. 2008; 46(11): 1745, 1786–8. PubMed Abstract \| Publisher Full Text\n\nRaether W, Hänel H: Epidemiology, clinical manifestations and diagnosis of zoonotic cestode infections: an update. Parasitol Res. 2003; 91(5): 412–38. PubMed Abstract \| Publisher Full Text\n\nSchroeder I, Altreuther G, Schimmel A, et al.: Efficacy of emodepside plus praziquantel tablets (Profender tablets for dogs) against mature and immature cestode infections in dogs. Parasitol Res. 2009; 105 Suppl 1: 31–8. PubMed Abstract \| Publisher Full Text\n\nCharles SD, Altreuther G, Reinemeyer CR, et al.: Evaluation of the efficacy of emodepside+praziquantel topical solution against cestode (Dipylidium caninum, Taenia taeniaeformis, and Echinococcus multilocularis) infections in cats. Parasitol Res. 2005; 97 Suppl 1: S33–40. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59825", "date": "03 Mar 2020", "name": "Alessandro Crimi", "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 article describes a case of Dipylidium caninum on a pregnant woman. The case is interesting as this is indeed a rare event.\nThere are however a few things which should be improved in the paper. Those have to be discussed.\nThe case report describes the inspection of a woman in 2013. Is this a typo? If it is not a typo. It is not clear why the case is reported now after 7 years. The situation of the parasites can have changed a lot. The tapeworm could have evolved, the environment can be different now, the cures could not be effective anymore…. This is a possible limitation that should be reported.\n\nGiven the description of the case it seems a very risky transmission with minimal contact (“the woman had no contact with dogs and cats but was just in areas where they were”). Can you compare the risk of transmission of dipylidiumto the taxoplasma gondii or similar hazards in the same area? e.g. in Kowofie et al. 20161.\n\nIt is not clear what are your perspectives and the lessons learned by the case. Do you suggest future policies to avoid contacts with dogs and cats? Do you suggest to reinforce stool analysis in case of reported vomiting? Do you suggest those to be applied to pregnant women or to the population in general reporting symptoms? Or since the case was treated so simply it is not a big deal? Then why reporting it?\n\nIn previous studies, including one of the reviewer (Amoah et al. 20162) women in rural areas avoid antenatal cares for series of reasons. Do you have updated statistics? Even just for the area of the reported case.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "67767", "date": "24 Aug 2020", "name": "Matthew T Brewer", "expertise": [ "Reviewer Expertise Parasitology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a case of D. caninum in a pregnant woman. Although the report in a pregnant woman appears to be slightly novel, the finding of D. caninum in a human is not unusual. There is no direct evidence (images, DNA sequences, histology, etc) presented.\n\n“Most of the cases ….are likely kissed or licked by pets”. This is an incorrect. The definitive host has to ingest the flea intermediate host, so licking/kissing doesn’t lead to to transmission.\n\nThe case was from 2013. It is odd that it took 7 years to be reported, which makes me somewhat skeptical of this report. Especially since there isn’t even an image of the egg. In general, there should be some evidence for the case report - images, genetic sequences, etc.\n\nIt is rare for cats to have obstruction, seizures, and convulsions. In animals, it is usually asymptomatic except for perhaps mild enteric signs and/or anal pruritis.\n\n“Diphylidiasis” should be dipylidiasis\n\nSince the authors suggest that the finding in a pregnant woman is novel, they should point out why it is especially important in this demographic.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/8-857
https://f1000research.com/articles/8-853/v1	12 Jun 19	{ "type": "Case Report", "title": "Case Report: Coronary artery stent infection with mycotic aneurysm secondary to tricuspid valve infective endocarditis", "authors": [ "Mejdi Ben Messaoud", "Nidhal Bouchahda", "Marouane Mahjoub", "Badii Hmida", "Zohra Dridi", "Habib Gamra", "Nidhal Bouchahda", "Marouane Mahjoub", "Badii Hmida", "Zohra Dridi", "Habib Gamra" ], "abstract": "Coronary artery stent infection with mycotic aneurysm is a rare life-threatening complication following coronary angioplasty, usually requiring surgical intervention. Reaching and confirming the diagnosis remains the most challenging aspect of this complication. We describe an unusual case of bare metal stent infection and coronary artery aneurysm in the setting of tricuspid valve infective endocarditis, resulting in ST elevation myocardial infarction, with a favorable outcome after primary angioplasty and antibiotic therapy. In the current era of growth of coronary stent implantation, it’s important for clinicians to consider and to prevent such potentially fatal events. The diagnosis process remains difficult and requires the association of multiple clinical, biological and imaging parameters. Although treatment modalities tend to favor surgery, we showed that coronary angioplasty could be a successful alternative solution.", "keywords": [ "endocarditis", "stents", "infection", "coronary aneurysm", "acute coronary syndrome" ], "content": "Introduction\n\nInfectious complications following percutaneous cardiac interventions are known but not very common. Coronary stent implantation is associated with complications, though rare, such as stent infection or coronary aneurysms resulting from several mechanisms, and needing special investigations for their diagnosis and management1,2. Simultaneous occurrence of coronary stent infection and mycotic aneurysm has been described in a few cases as an unusual life-threatening complication following coronary angioplasty3. The majority of the few reported cases had an unfavorable outcome4, with the majority treated surgically.\n\nWe report a rare case of ST elevation myocardial infarction due to bare metal stent infection and coronary artery aneurysm in the setting of tricuspid valve infective endocarditis. The management and outcome of our patient was quite different from what was reported in the literature.\n\nIt is important for the interventional cardiologist, to remember that such complication might occur and should be prevented. If it does occur however, its treatment and outcome represent a challenge for clinicians.\n\n\nCase\n\nA 71-year-old North African retired male patient with a history of smoking (40 pack-years) and no known medical or surgical history, was referred to our center in October 2017, from the emergency department, for inferior ST elevation myocardial infarction with complete atrioventricular block. He underwent a successful primary angioplasty of the right coronary artery (RCA) with a bare metal stent (Figure 1). A temporary cardiac pacing via the right femoral vein was performed for 24 hours and the patient was discharged at day 5. Initial and controlled laboratory tests during hospitalization were normal except elevated troponin at admission. One week later, the patient was referred again to our center from the emergency department for persistent chest pain with a new right bundle branch block. An urgent coronary angiogram showed a thrombus with an aneurysm on the distal part of the RCA stent and a Thrombolysis In Myocardial Infarction (TIMI) 3 flow (normal flow) (Figure 2A and 2B). An additional angioplasty using a second bare metal stent overlapping with the previous one was successfully performed with exclusion of the aneurysm and disappearance of the thrombus. (Figure 2B and 2C). The initial physical exam in the coronary care unit immediately after angioplasty found a mild fever at 38°C with a systolic tricuspid regurgitation murmur. Echocardiography showed vegetation (17 × 14 mm) at the level of the tricuspid valve with a moderate tricuspid regurgitation and a moderate pericardial effusion (Figure 3A and 3B). Transesophageal echocardiography confirmed the same findings and ruled out a patent foramen ovale or any involvement of the other valves. Laboratory tests (which were normal at the previous admission) showed a marked elevation of white blood cells count (24,000 E/mm3, normal value < 11,800 E/mm3), C reactive protein (186 mg/L, normal value < 6 mg/L), and liver enzymes (Alanine transaminase (ALT) at 215 IU/L and Aspartate aminotransferase (AST) at 165 IU/L, normal value respectively < 35 IU/L and < 40 IU/L) associated with acute renal failure (serum creatinine at 148 mmol/L, normal value < 105 mmol/L) and hemolytic anemia (Hemoglobin 8 g/DL, normal value > 13 g/DL). Blood cultures were positive to negative coagulase Staphylococcus. The diagnosis of infective endocarditis of the tricuspid valve with RCA mycoticaneurysm at the site of stent implantation was strongly suspected. Cardiac CT scan confirmed the vegetation in the right ventricle with hematoma around the RCA and pericardial effusion (Figure 3C). It also showed a mycotic aneurysm of the RCA which was excluded by the overlapping stent (Figure 3D). Following a thorough search for the underlying cause, we concluded that the temporary cardiac pacing was the most likely origin of the tricuspid valve infective endocarditis. Antibiotic therapy (vancomycin 30 mg/Kg/day i.v. in 2 doses, rifampicin 900 mg orally in 3 divided doses and gentamycin 3 mg/Kg i.v once daily) was provided resulting in total regression of the infection symptoms, the tricuspid vegetation and the pericardial effusion. The patient was discharged from the hospital after six weeks of antibiotic therapy. The patient has completed one-year of follow-up with repeated echocardiography and blood tests, all of which were ordinary.\n\nCoronary angiogram images before (A) and after (B) the first primary angioplasty of the right coronary artery.\n\nCoronary angiogram images of the second primary angioplasty: A and B show the intrastent thrombus with mycotic aneurysm of the right coronary artery. C and D show the right coronary artery angioplasty with an overlapping bare metal stent.\n\nImaging showing tricuspid valve vegetation (arrow) with hematoma around the right coronary artery (arrowhead) and pericardial effusion in transthoracic echocardiography (A and B) and cardiac computed tomography scan (C). D: computed tomography coronary angiography showing the right coronary artery aneurysm excluded by the overlapping stent.\n\n\nDiscussion\n\nStent infection can occur early (less than 10 days) or late (10 days or longer) following coronary angioplasty4 and is frequently diagnosed in the context of an acute coronary syndrome due to stent thrombosis or coronary artery aneurysm. The diagnosis of coronary stent infection is confirmed based on the criteria proposed by Dieter1. The diagnosis is definitive when confirmed by autopsy or surgical material examination if at least three of the following criteria are present: coronary stent implantation during the last 4 weeks; repeated interventions through the same arterial sheath; fever > 38°C, documented bacteremia, leukocytosis without evident etiology; acute coronary syndrome; or positive cardiac imaging. Mycotic coronary aneurysms may result from different mechanisms including direct bacterial invasion in the setting of infective endocarditis, injury caused by immune complexes, arterial trauma and vasa vasorum embolic occlusion. Other mechanisms such as immuno-compromised status and congenital cardiovascular defects might be involved2. Following testing for all these mechanisms in our patient we strongly suspected the infective endocarditis as the main etiology of the right coronary stent mycotic aneurysm. The majority of the similar reported cases were associated with a fatal outcome4. In few cases, like in ours, peri-coronary hematoma with pericardial effusion can be present and it may be due to the coronary aneurysm leak or rupture5. Irrespective of time of presentation, the majority of stent infection with coronary aneurysm must be treated by surgical extraction of the stent with aneurysm repair when indicated and with simultaneous intravenous antibiotic therapy for at least 4 weeks6. In our case, the follow up at 1 year didn’t show any signs of relapse. Consequently, the patient was not referred to surgery.\n\nOne limitation of this report relates to the mycotic nature of the stent aneurysm which can only be confirmed by tissue analysis. This was not possible since the patient did not have surgery nor a postmortem autopsy. However, the diagnosis was highly probable based on the clinical, biological and imaging parameters.\n\nIn conclusion, coronary stent infection in the setting of infective endocarditis is rare but must be considered whenever a patient develops fever and chest pain after stent implantation. Treatment modalities tend to favor surgery, but in our case, we showed that coronary angioplasty and prolonged antibiotic therapy may be sufficient. To prevent such complications, adherence to aseptic precautions and treatment of pre-existing infections are of paramount importance.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient/parent/guardian/relative of the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nDieter RS: Coronary artery stent infection. Clin Cardiol. 2000; 23(11): 808–810. PubMed Abstract \| Publisher Full Text\n\nWeinstein L, Schlesinger JJ: Pathoanatomic, pathophysiologic and clinical correlations in endocarditis (second of two parts). N Engl J Med. 1974; 291(21): 1122–1126. PubMed Abstract \| Publisher Full Text\n\nMatsumoto M, Konishi Y, Miwa S, et al.: Mycotic aneurysm of the left coronary artery. Ann Thorac Surg. 1998; 65(3): 841–2. PubMed Abstract \| Publisher Full Text\n\nElieson M, Mixon T, Carpenter J: Coronary stent infections: a case report and literature review. Tex Heart Inst J. 2012; 39(6): 884–9. PubMed Abstract \| Free Full Text\n\nRoubelakis A, Rawlins J, Baliulis G, et al.: Coronary artery rupture caused by stent infection: a rare complication. Circulation. 2015; 131(14): 1302–1303. PubMed Abstract \| Publisher Full Text\n\nSingh H, Singh C, Aggarwal N, et al.: Mycotic aneurysm of left anterior descending artery after sirolimus-eluting stent implantation: a case report. Cathet Cardiovasc Interv. 2005; 65(2): 282–285. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49816", "date": "10 Jul 2019", "name": "Nicolas Amabile", "expertise": [ "Reviewer Expertise interventional cardiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, Ben Messaoud and colleagues reported a very unusual case of a patient who developed a subacute stent thrombosis in the setting of a tricuspid acute endocarditis. The ST was associated with an image of aneurysm on the distal edge of the stent. Although there are other causes of coronary artery aneurysm (including iatrogenic media disruption, stent fracture or severe stent malapposition) that could appear after initial thrombus dissolution, the authors concluded to a mycotic aneurysm. This conclusion makes sense regarding the clinical context, CT scan data and subsequent evolution following antibiotics therapy. The main limitation of the report is the lack of pathology analyses and was acknowledged by the authors.\n\nThe manuscript in this current version raises the following issues:\nWhat was the antiplatelet drug regimen of the patient after the first STEMI ? What about the patient compliance?\n\nThe CT scan initial data could be better explained and a cross sectional image of the RCA showing the hematoma might be a nice addition.\n\nDo the authors have a 1 year CT scan or angio to illustrate the aneurysm disappearance (or no)?\n\nWhat is the authors opinion about implanting a new device in an actively infected stent?\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": "4744", "date": "11 Jul 2019", "name": "Mejdi Ben Messaoud", "role": "Author Response", "response": "I would like to thank Dr Amabile for his precious comments. The answers for his questions are as follows: The antiplatelet therapy included aspirin 100 mg once a day and Clopidogrel 75 mg once a day. There was no history of DAPT discontinuation. I agree that a cross sectional image of the RCA showing the hematoma might be a nice addition. No CT scan was performed after 1-year follow up. The follow up was based on clinical exam, biological tests and echocardiography. We think that implanting a new device in an actively infected stent is not recommended, but in our case, we have performed an ad-hoc angioplasty because the diagnosis of stent infection was not confirmed." } ] }, { "id": "49820", "date": "15 Oct 2019", "name": "Marouane Boukhris", "expertise": [ "Reviewer Expertise Interventional cardiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the current manuscript, the authors reported a rare infective complication following percutaneous coronary intervention. They underlined the difficulty of the diagnosis, particularly when neither surgery nor autopsy is performed. Generally, the manuscript is well written and the topic is quite interesting for the reader. The paper seems suitable for indexing in its actual version.\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/8-853
https://f1000research.com/articles/8-852/v1	12 Jun 19	{ "type": "Research Article", "title": "Diversity issues in Nigeria’s healthcare sector: implications on organizational commitment. A cross-sectional study", "authors": [ "Ayodotun Stephen Ibidunni", "Tomike Olawande", "Maxwell Olokundun", "Charles Iruonagbe", "Iyanu Adelekan", "Tomike Olawande", "Maxwell Olokundun", "Charles Iruonagbe", "Iyanu Adelekan" ], "abstract": "Background: Workplace diversity is increasingly gaining the attention of healthcare organizations, especially in developing countries like Nigeria. However, little is understood from existing literature about how workforce diversity affects employees’ satisfaction and organisational commitment in the workplace. Consequently, this paper showed the direct and mediating relationships between diversity of workforce, job satisfaction and employee commitment to the organization. Methods: Copies of the structured questionnaire have been given to 133 public healthcare employees in Nigeria’s Ministry of Health in Lagos state. Statistical analysis for the study included descriptive measures and multi-variate analysis, using structural equation modelling. Results: Outcomes from statistical analysis supports direct and mediating relationships between the research variables. Gender and ethical diversity had significant influences on job satisfaction at r = 0.35 (p < 0.05) and r = 0.28 (p < 0.05) respectively. The following mediating relationships were also statistically confirmed: job satisfaction related with affective commitment (r = 0.41, p < 0.05) and normative commitment (r = 0.26, p < 0.05). Conclusions: Based on the results of the statistical analysis, the study concludes that there is a relationship between diversity of employees and job satisfaction, diversity of employees and organizational commitment and the influence of work satisfaction on organizational commitment.", "keywords": [ "Workforce diversity", "Job satisfaction", "Employee commitment", "Public healthcare", "Diversity management" ], "content": "Introduction\n\nWorkforce diversity has a significant impact on the work behaviour and attitudes that employees exhibit in the work environment1. Diversity of employees depicts a scenario in which different people bring their unique backgrounds, perspectives, values and benefits to the organization2.\n\nAssessing diversity among employees is strategic to effectively managing the organization’s human resources and can improve the organizational commitment and satisfaction of employees3. Furthermore, diversity in the workforce improves better decision-making, greater creativity, innovation and greater competitiveness4–5. Workforce diversity has grown from focusing solely on human demographic characteristics; like race, gender and age, to a much broader meaning that encompasses the whole range of human, physical and cultural differences6, and this could influence their levels of job satisfaction.\n\nIn the work of Jex7, job satisfaction is described as the positive affection of an employee towards the job. According to Shuck et al.8, employee satisfaction itself has a direct and indirect influence on individual and organizational performance, including the commitment of employees to work. Job satisfaction, for example, is the best predictor of employee turnover. More so, having a committed workforce is important for achieving organisational performance. Therefore, it is important to produce a committed workforce that would show organisational commitment.\n\nToday's workplace consists of different employees with unique and varied features9,10. Although, existing literature have discussed workforce components as a determinant of employee commitment11. However, the current literature does not sufficiently explained the employees feelings about diversity in their working environment, and whether diversity in the workforce results in dissatisfaction or lack of engagement, especially within the public health sector of developing economies like Nigeria. Despite that most researchers, such as Osibanjo et al.12 have been focusing on explaining concepts that enhance understanding of workforce diversity, little is understood about how workforce diversity affects employees’ satisfaction and organisational commitment in the workplace. Studies on the influence of diversity in the workforce on the satisfaction of employees and the organizational commitment in the public health sector in Nigeria are rare in existing literature13. This paper therefore aims to examine the influence of diversity in the workforce on organizational commitment and employee satisfaction among selected public health workers in Nigeria, based on the following four hypotheses.\n\nH1: Employee diversity significantly influences employees’ affective commitment\n\nH2: Employee diversity significantly influences employees’ continuance commitment\n\nH3: Employee diversity significantly influences employees’ normative commitment\n\nH4: Job satisfaction moderates the relationship between workforce diversity and organizational commitment (affective, continuance and normative)\n\n\nMethods\n\nA survey method was used for data collection.\n\nThis research involved 133 public health workers from the Lagos State Ministry of Health in Nigeria and was executed between the periods of February 2017 and April 2019. This sample size was determined from a population of 200 public health workers, based on Yamane14 sample size determination formula. Thus,\n\nThe formula\n\nn=N/(1+N(e)2)\n\nwhere\n\nn= sample size\n\ne= sampling error (0.05)\n\nN= the population (that is 200 employees in the healthcare location)\n\nn = 200 / (1+200(0.05)2)\n\nn =200/ 1+ 200 (0.0025)\n\nn =200/ 1+ 0.5\n\nn =200/ 1.5\n\nn = 133.33 (approximately 133)\n\nn = 133\n\nPublic healthcare employees in Nigeria’s Ministry of Health, are important for this research because of the increased awareness of the diverse workforce of the Ministry. Hence, it is imperative that such diversity is properly managed in ways that avoid negative impact on employee satisfaction and commitment to the organization15,16. Furthermore, due to the important roles that the Health sector undertake for the population and the overall well- being of any country, this research is considered essential to maintain employee interest and motivation in the provision of quality services17. Simple random sampling technique was adopted. This technique allowed every member of the population to be a respondent by selecting the respondents without any form of partiality. Thus, given the total population if public health workers in the Lagos State Ministry of Health, the 133 respondents were selected at random without any form of bias and were administered the copies of the questionnaire during working hours. Employees who turned down the proposal to participate in the survey were respectfully eliminated from the research process, and a replacement was determined by the same random selection process. The following inclusion and exclusion criteria applied:\n\nInclusion criteria:\n\n• Participants had to be employees of the organisation\n\n• Participants must be literate, able to read and write English\n\n• Participants must be accessible\n\nExclusion criteria:\n\n• Members of casual staff\n\nThe copies of questionnaire were self-administered to respondents and retrieved within the space of eight weeks, based on the request of the respondents.\n\nData was captured using a questionnaire developed for this study based on evidence from the literature (see extended data18). Workforce diversity (including gender, education, religion, ethnicity, experience, income and position) items were developed based on Glazer et al.19, work satisfaction (described as by the extent to which employees felt positive affection towards their job) items based on Jensen et al.20 were developed and workforce commitments (including affective, continuance and normative) items based on Changa et al.21 and22 Hanaysha et al.22 were developed.\n\nThe questionnaire was administered to respondents based on their willingness to participate in the research exercise. The participants in the study were also assured of confidentiality and anonymity, such as not reflecting their names in the questionnaire. The study received verbal approval for execution as a satisfactory requirement from the organization and the employees.\n\nIn this research, internal consistency method, using Cronbach alpha measurement was adopted to ensure reliability of the research items. It is widely accepted that the score of 0.7 above indicates the reliability of the instrument. The SPSS (Social Science Statistical Package) was used to test the reliability of the research instrument. The reliability statistics that was obtained for the instruments used for this study was 0.747. Instrument validity was ascertained through content validity.\n\nThe statistical analysis for this study was performed using SPSS software (version 22) and AMOS (version 23). The analysis of data was carried out using descriptive statistics and structural equation modelling (SEM) to show the relationships between workforce diversity, job satisfaction and organizational commitment.\n\n\nResults\n\nIn terms of demographics of the respondents, 64 (48.1%) were male while 69 (51.9%) were female. The age distribution among respondents was as follows: the majority of respondents (68) were between 18 and 34 years of age, 45 respondents between 35 and 44 years of age, 13 respondents were between 45 and 54, and 7 were 55 or above years (Table 1 and underlying data23).\n\nFigure 1 shows the model for analysis of multi-variate relationships between diversity of workforce, job satisfaction and employee commitment. The statistical measurement was considered fit based on the following estimates: Chi-square / degree of freedom (Cmin / df) = 1.603, goodness of fit index (GFI)= 0.950, normal fit index (NFI)= 0.892, comparative fit index (CFI)= 0.952, root mean square approximation error (RMSEA)= 0.068. These estimates are considered to be fit based on existing studies24,25.\n\nAnalyses revealed varying levels of direct and mediating relationships among the research variables. Specifically, gender and ethical diversity had significant influences on job satisfaction at r = 0.35 (p < 0.05) and r = 0.28 (p < 0.05) respectively. Direct relationships were also statistically established between workforce diversity and organizational commitment in the following ways: gender diversity and affective commitment (r = 0.26, p < 0.05), normative commitment (r = 0.05, p < 0.05); education diversity and normative commitment (r = 0.12); experience diversity and continuance commitment (r = 0.20, p < 0.05). A direct inverse relationship was established between religion diversity and continuance commitment (r = -0.14, p < 0.05). The following mediating relationships were also statistically confirmed: job satisfaction related with affective commitment (r = 0.41, p < 0.05) and normative commitment (r = 0.26, p < 0.05). The results of these data support existing research results21,26.\n\n\nDiscussion and Conclusion\n\nResearchers like Ashikali et al.27, Ibidunni et al.26 and Hanaysha et al.28 demonstrated that diversity in the workforce promotes creativity, innovative problem solving and productivity. Also, Moon28 confirmed that large organizations use various teams intentionally to solve problems and employed people from a diversity races, ethnicities and experience to shape perspectives in the organization29,30.\n\nAlthough arguments for diversity are naturally attractive and somewhat popular, there is little empirical evidence that diversity in the workforce can be used to increase employee engagement, especially in developing economies such as Nigeria. Whether employee diversity influences organizational engagement is an empirical issue that has not been properly tested in Nigeria in the public sector context. This study therefore contains evidence to fill this gap. The results of this study show that gender is the most sensitive factor in the diversity that influences Nigeria's commitment to public health workers. Gender diversity is associated with the affective and normative aspects of the commitment of employees31,32. These two dimensions of commitment reflect the degree to which employees are emotionally committed to their productivity at work26,33. The promotion of gender equality at work must therefore be seen as a strategic way to increase the commitment of public health workers in Nigeria.\n\nOur results demonstrate that the diversity of the workforce has a significant impact on the satisfaction and commitment of Nigerian public health professionals. Based on the results of the statistical analysis, the study concludes that there is a relationship between diversity of employees and job satisfaction, diversity of employees and organizational commitment and the influence of work satisfaction on organizational commitment. Therefore, this study has important implications for directing policymakers to ensure that the demographic diversity among public health workers is treated as a strategic part of employee selection. This in turn significantly influences their levels of job satisfaction and commitment to the organization.\n\n\nData availability\n\nFigshare: ADELEKAN SPSS.sav, https://doi.org/10.6084/m9.figshare.8144498.v123\n\nThis project contains the following underlying data:\n\nADELEKAN SPSS.sav (SPSS spreadsheet of primary data collected to establish the relationships between workforce diversity, job satisfaction and organisational commitment of public health workers in Lagos state, Nigeria)\n\nFigshare: Questionnaire. https://doi.org/10.6084/m9.figshare.8144564.v118\n\nThis project contains the following extended data:\n\nRESEARCH QUESTIONNAIRE.pdf (Study questionnaire)\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\nAuthors of this research work would like to appreciate Covenant University Management for providing sponsorship to the publication of the research in this journal.\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\nMcKay PF, Avery DR, Morris MA: Mean Racial-Ethnic Differences In Employee Sales Performance: The Moderating Role Of Diversity Climate. Pers Psychol. 2008; 61(2): 349–374. Publisher Full Text\n\nOtike W, Messah B, Mwaleka K: Effects of Workforce Diversity on Organizational Effectiveness: A Case Study On Kenya Commercial Bank Ltd. Journal Of Business And Management. 2010. Reference Source\n\nBlack & Enterprise: “Managing a multicultural workforce. Black Enterprise Magazine (July).Canada”, Equal Opportunities International. 2001; 15(5): 1–27.\n\nGupta R: Workforce Diversity and Organizational Performance. International Journal of Business Management Invention. 2013; 2(6): 36–44. Reference Source\n\nDaft RL, Kendrick M, Natalia V: Management. Hampshire: South Western Cengage Learning. 2010. Reference Source\n\nJayne M, Dipboye R: Leveraging diversity to improve business performance: Research findings and recommendations for organizations. J Hum Resour Manag. 2004; 43(4): 409–424. Publisher Full Text\n\nJex SM: Organizational psychology: A scientist-practitioner approach. New York, NY:John Wiley & Sons, Inc. 2002. Reference Source\n\nShuck B, Collins JC, Rocco TS, et al.: Deconstructing the Privilege and Power of Employee Engagement: Issues of Inequality for Management and Human Resource Development. Hum Resour Dev Rev. 2016; 15(2): 208–229. Publisher Full Text\n\nGoby VP, Nickerson C, David E: Interpersonal communication and diversity climate: promoting workforce localization in the UAE. Int J Organ Anal. 2015; 23(3): 364–377. Publisher Full Text\n\nSingh SK: Territoriality, task performance, and workplace deviance: Empirical evidence on role of knowledge hiding. J Bus Res. 2019; 97: 10–19. Publisher Full Text\n\nSchilpzand P, De Pater IE, Erez A: Workplace incivility: A review of the literature and agenda for future research. J Organ Behav. 2016; 37(S1): 57–88. Publisher Full Text\n\nOsibanjo AO, Abiodun AJ, Adeniji AA: Impact of job environment on job satisfaction & commitment among Nigerian nurses. Proceedings of the 22nd International Business Information Management Association Conference, 2013; 3: 1743–1752. Reference Source\n\nWatson WE, Johnson L, Zgourides GD: The influence of ethnic diversity on leadership, group process, and performance: An examination of learning teams. Int J Intercult Relat. 2002; 26(1): 1–16. Publisher Full Text\n\nYamane T: Statistics, An Introductory Analysis. 2nd Ed., New York: Harper and Row. 1967. Reference Source\n\nAbiodun AJ, Ibidunni AS, Kehinde OJ: Demographic Determinants of Communication and Information Technology Appreciation and Usage among Health Care Professionals. Int J Health Econ. 2015; (5): 5–19. Reference Source\n\nFalola HO, Olokundun MA, Salau OP, et al.: Data article on the effect of work engagement strategies on faculty staff behavioural outcomes in private universities. Data Brief. 2018; 18: 1383–1387. Publisher Full Text\n\nAyeni AW, Iyiola OO, Ogunnaike OO, et al.: Globalisation and Ebola disease: Implications for business activities in Nigeria. Afr J Bus Manage. 2017; 11(3): 47–56. Publisher Full Text\n\nIbidunni AS: Questionnaire. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.8144564.v1\n\nGlazer G, Tobias B, Mentzel T: Increasing healthcare workforce diversity: Urban universities as catalysts for change. J Prof Nurs. 2018; 34(4): 239–244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJensen KW, Liu Y, Schøtt T: Entrepreneurs’ innovation bringing job satisfaction, work-family balance, and life satisfaction: In China and around the world. Int J Innov Stud. 2017; 1(4): 193–206. Publisher Full Text\n\nChanga E, Chinb H: Signaling or experiencing: Commitment HRM effects on recruitment and employees' online ratings. J Bus Res. 2018; 84: 175–185. Publisher Full Text\n\nHanaysha J: Examining the Effects of Employee Empowerment, Teamwork, and Employee Training on Organizational Commitment. Procedia Soc Behav Sci. 2016; 229: 298–306. Publisher Full Text\n\nIbidunni AS: ADELEKAN SPSS.sav. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8144498.v1\n\nHipp JR, Bollen KA: Model Fit in Structural Equation Models with Censored, Ordinal, and Dichotomous variables: Testing Vanishing Tetrads. Sociol Methodol. 2003; 33: 267–305. Publisher Full Text\n\nSchumacker RE, Lomax RG: A beginner’s guide to structural equation modelling. (3rd ed.). New York: Routledge. 2010. Reference Source\n\nIbidunni AS, Falola HO, Ibidunni OM, et al.: Workforce diversity among public healthcare workers in Nigeria: Implications on job satisfaction and organisational commitment. Data Brief. 2018; 18: 1047–1053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshikali T, Groeneveld S: Diversity Management in Public Organizations and Its Effect on Employees’ Affective Commitment: The Role of Transformational Leadership and the Inclusiveness of the Organizational Culture. Rev Public Pers Adm. 2015; 35(2): 146–168. Publisher Full Text\n\nMoon KK: Examining the Relationships Between Diversity and Work Behaviors in U.S. Federal Agencies: Does Inclusive Management Make a Difference? Rev Public Pers Adm. 2018; 38(2): 218–247. Publisher Full Text\n\nBransford JD, Brown AL, Cocking RR, (Eds): How people learn: Brain, Mind, Experience and School. Washington D.C: National Academy Press. 2000. Publisher Full Text\n\nKreitner R, Kinichi A: Organizational Behaviour. Boston: McGraw-Hill. 2004. Reference Source\n\nChoi S: Workforce Diversity and Job Satisfaction of the Majority and the Minority: Analyzing the Asymmetrical Effects of Relational Demography on Whites and Racial/ Ethnic Minorities. Rev Public Pers Adm. 2017; 37(1): 84–107. Publisher Full Text\n\nOyinlade AO: Relations of Job Structure to Affective Organizational Commitment. J Hum Resour Manag Labor Stud. 2018; 6(1): 13–32. Reference Source\n\nMessner W: The role of gender in building organisational commitment in India’s services sourcing industry. IIMB Management Review. 2017; 29(3): 188–202. Publisher Full Text" }	[ { "id": "77553", "date": "19 Jan 2021", "name": "Henry Inegbedion", "expertise": [ "Reviewer Expertise Operations Management", "Quantitative Analysis", "Marketing and Financial Economics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research problem is very relevant and timely, given the challenges of the health sector globally amidst the COVID-19 pandemic. The authors did well to articulate the problem convincingly. However, there are some issues that require adequate attention in order to enhance the quality of the paper:\nThe article needs to be thoroughly edited to take care of the grammatical errors.\n\nThe paper is not underpinned by any theory. This is a significant omission since some of the underlying concepts in the research problem have theoretical basis that can be usefully employed.\n\nThe authors did not develop the hypotheses formulated. How did they come about the hypotheses? The absence of hypotheses development is a consequence of the weak empirical review.\n\nThe methodology section is good but the authors need to indicate the basis of randomization in sample selection as well as how content validity was employed in the validation of the research instrument. Lastly, they should justify the application of the structural equation model, provide a mathematical formulation of the model and segregate the measurement model from the latent component.\n\nIn the interpretation of results, the authors should check gender diversity and normative commitment (r =0.05, p < 0.05) as well as the statement: \"A direct inverse relationship was established between religion diversity and continuance commitment\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "74647", "date": "21 Jan 2021", "name": "Joonghak Lee", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for your interesting article.\nI have a few comments on your literature review and method:\nFirst, it would be much better cite recent diversity articles. Also, there should be more studies about the relationship between dependent variables and independent variables.\n\nSecond, even if you use cross-sectional data, you don't mention anything about its limitation such as the Common Method bias. The authors should try to analyze CFA or Harman's single factor analysis to minimize reviewer's concern.\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/8-852
https://f1000research.com/articles/8-851/v1	12 Jun 19	{ "type": "Research Article", "title": "Measuring bias, burden and conservatism in research funding processes", "authors": [ "Susan Guthrie", "Daniela Rodriguez Rincon", "Gordon McInroy", "Becky Ioppolo", "Salil Gunashekar", "Daniela Rodriguez Rincon", "Gordon McInroy", "Becky Ioppolo", "Salil Gunashekar" ], "abstract": "Background: Grant funding allocation is a complex process that in most cases relies on peer review. A recent study identified a number of challenges associated with the use of peer review in the evaluation of grant proposals. Three important issues identified were bias, burden, and conservatism, and the work concluded that further experimentation and measurement is needed to assess the performance of funding processes. Methods: We have conducted a review of international practice in the evaluation and improvement of grant funding processes in relation to bias, burden and conservatism, based on a rapid evidence assessment and interviews with research funding agencies. Results: The evidence gathered suggests that efforts so far to measure these characteristics systematically by funders have been limited. However, there are some examples of measures and approaches which could be developed and more widely applied. Conclusions: The majority of the literature focuses primarily on the application and assessment process, whereas burden, bias and conservatism can emerge as challenges at many wider stages in the development and implementation of a grant funding scheme. In response to this we set out a wider conceptualisation of the ways in which this could emerge across the funding process.", "keywords": [ "Bias", "burden", "conservatism", "innovation", "creativity", "peer review", "grant funding" ], "content": "Introduction\n\nPeer review is a core part of the academic research process, with the majority of academic research funding allocated through peer review. In a recent review (Guthrie et al., 2018), some of the potential limitations and outcomes of the use of peer review to allocate research funding were explored, with a key finding of that work being that there is a need for further experimentation and evaluation in relation to peer review and the grant award process more widely. However, measuring the performance of the funding allocation processes can be challenging, and there is a need to better share learning and approaches. This paper aims to address this gap by providing a review of existing practice in the measurement of research funding processes in relation to three of the main challenges identified by Guthrie et al. (2018):\n\n- Level of burden\n\n- Evidence of bias\n\n- Supporting innovative and creative research\n\nThe intention of this work is to provide a review of ideas and approaches that funders can use to better analyse their own funding processes and to help facilitate a more open and analytical review of funding systems. Through our interviews with funders, we also explored current practice internationally in attempting to reduce burden and bias and to facilitate innovation and creativity in research.\n\n\nMethods\n\nWe undertook a Rapid Evidence Assessment (REA) that built on previous work—such as that by Guthrie et al. (2018) —encompassing methods for evaluating programs, the challenges faced in evaluation, issues associated with research evaluation and the importance of responsible metrics. We focused specifically on metrics and measurement approaches that address bias, burden and conservatism. We restricted our search to literature in English from the 10 years between 2008 and 2018 to ensure we focused on the latest developments in the field and current best practice. We covered academic literature in Scopus as well as grey literature, e.g. policy reports and studies, government documents and news articles.\n\nWe identified relevant literature through three routes:\n\n1. Academic literature search: Scopus search using the search terms in Table 1 for publications from 2008 onwards. To identify literature that focused on bias, burden and conservatism, we operationalised these search strings as follows: [Group 1 AND Group 2 AND (Group 3 OR Group 4 OR Group 5 OR Group 6 OR Group 7)].\n\n2. Grey literature search: search on the websites of the funding bodies considered in this study (Table 2)\n\n3. Snowballing: Snowballing refers to the continuous, recursive process of gathering and searching for references from within the bibliographies of the shortlisted articles. We performed snowballing from the reference lists of publications identified following screening.\n\nUsing the search strings described above, the Scopus database yielded 1,741 results. We performed an initial test of our strategy by checking that specific key papers we were already aware of appeared in the results, for example Guthrie et al. (2018). Once satisfied the search strategy was performing effectively, we implemented a filtering process to determine the inclusion or exclusion of articles based on their relevance to address the primary objectives of this task as set out in Figure 1.\n\nData extraction was performed using a data extraction template—a pre-determined coding framework based on the study aims (i.e. bias, burden, and conservatism). The headers of this template against which data was extracted for each article (where available) were:\n\nResearcher extracting data (initials)\n\nSource (author, date)\n\nTitle\n\nYear of publication\n\nURL\n\nType of document (journal article, review, grey report, book, policy, working paper, etc)\n\nObjectives (aims of the work)\n\nArea of peer review (journals, grants, other)\n\nEvaluation framework or model (to evaluate funding program)\n\nEvidence on and measures of burden (on researchers, institutions, funding bodies)\n\nEvidence on and measures of bias (gender, career stage, research type, institution)\n\nEvidence on and measures of innovation\n\nDatasets and collection (any datasets used for evaluation purposes or information on data collection)\n\nMetrics and indicators (any specified metrics used for evaluation)\n\nChallenges (any identified challenges for evaluations)\n\nStrengths and weaknesses (of the study)\n\nQuality of study design and conduct (if appropriate assign red, amber, or green)\n\nStrength and generalisability of the findings (assign red, amber, or green)\n\nThree researchers performed the full extraction of 100 articles in parallel. During this process, each researcher was instructed to add key references to a ‘snowballing database’. The snowballing database was populated with 15 articles, which were passed through the filtering processes described above, yielding an additional eight papers that were fully extracted. We also considered additional articles using a combination of targeted web searches and suggestions from our key informant interviews. These methods yielded an additional 18 articles that were included in our REA.\n\nWe conducted key informant interviews with one representative from each research funding organisation in Table 2 in order to understand how evaluation methods are employed in practice and to explore evaluation approaches that may not be documented in the literature. We identified respondents with relevant expertise at key biomedical and health research funders internationally and contacted them by email to request their participation. We focused on developed research systems that may be comparable with the Australian system, primarily in Europe and North America. We also interviewed researchers working on the analysis of peer review and grant funding approaches and their challenges. Initially 12 individuals were contacted. Of those, 6 agreed to participate; 5 did not respond to our request; 1 declined to participate; 2 further identified colleagues to participate in their place, who were contacted by email and in both cases accepted.\n\nInterviews were conducted by telephone and lasted for approximately one hour. Interviews were recorded and field notes taken. One interview was conducted per participant. Interviews were conducted following a semi-structured protocol (see Table 3) to enable consistent evidence collection while providing the opportunity to explore emerging issues. As the interviews were designed to be semi-structured, we encouraged the interviewees to explore areas they thought were important that may not have been directly covered in our interview protocol. To protect the anonymity of the interviewees, the analysis that we report does not make any specific reference to individuals; we use the interview identifiers INT01, INT02, etc. to make references to specific interviews in our analysis.\n\nThe analysis took a framework analytic approach, aiming to capture information on processes and metrics used in practice across organisations in relation to the aims of this study to identify how bias, burden and innovation in funding process can be measured. Data from each interview was coded into an excel template by each individual conducting interviews (GM, DRR), with one row per interview (and hence organisation). The column headers were as follows:\n\nOrganisation name\n\nAims/structure of funding programme\n\nApplication process\n\nReview process\n\nBurden\n\nBias\n\nInnovation\n\nEvaluation method\n\nMetrics used for evaluation\n\nChallenges\n\nStrengths\n\nWeaknesses\n\nAnalysis was primarily focused on capturing information on practice at each of these organisations to provide a picture of the methods currently being used by research funding organisations to measure and to alleviate burden, bias and conservatism in peer review-based funding processes. However, we also reviewed evidence on challenges, strengths and weaknesses to identify any information to inform our wider analysis and discussion.\n\nThis study was recommended for exemption by the RAND Human Subjects Protection Committee. Participant consent was obtained orally at the start of each interview. The precise detail of consent sought is set out in the interview protocol (Table 3).\n\n\nResults\n\nRobustness of procedure and efficiency of funding distribution are the two pillars supporting the legitimacy of peer review (Gurwitz et al., 2014), which has been described as a cornerstone of the scientific method (Tomkins et al., 2017) and the backbone of modern science (Tamblyn et al., 2018). A robust peer review process must be fair and objective in the distribution of grants. However, an increasing number of studies suggest that systematic bias occurs in a range of forms across grant peer review processes.\n\nWhile there are an increasing number of studies examining bias in grant peer review, there is still deemed to be a lack of comparable, quantitative studies in the area (Bromham et al., 2016; Gurwitz et al., 2014; Guthrie et al., 2018). The main body of work has been performed by analysing historic data made available by funding agencies to academic researchers, though funding bodies themselves have undertaken work in the area (DFG, 2016; Ranga et al., 2012). A challenge in identifying and evaluating sources of bias is the lack of generalisability of findings, as funding programs have highly variable structures and funding bodies collect and make available different datasets. Table 4 lists the measurement approaches employed in the literature, indicates which areas of potential bias were explored and provides key references. A table listing each item identified during the literature review is available as Underlying data (Guthrie, 2019).\n\n1 AQuAS is not a funding agency. AQuAs is a non-profit public assessment agency of the Catalan Ministry of Health (Spain).\n\nApplicant characteristics. Applicant characteristics collected during grant application processes may include gender, age, race, ethnicity and nationality. While reviewers do not usually see information about all characteristics, for instance race and ethnicity (Erickson, 2011), it may be apparent due to name, affiliation or prior knowledge.\n\nGender bias has been the primary area of study within applicant characteristics, perhaps having gained significant visibility in an early study that showed that females needed to be 2.5-fold more productive to achieve the same scores as males in the Swedish Medical Research Council’s peer review process (Wennerås & Wold, 1997).\n\nFollowing this initial study, gender bias has been explored in several different countries. In The Netherlands, researchers funded by The Netherlands Organisation for Scientific Research (NWO) examined 2,823 applications between 2010 and 2012 from early career scientists, analysed gender as a statistical predictor of funding rate, and examined the success rate throughout the process (application, pre-selection, interview, award) (Van Der Lee et al., 2015) The authors found that there was a gender disparity with males receiving higher scores in ‘quality of researcher’ evaluations but not ‘quality of proposal’ evaluations, particularly in disciplines with equal gender distribution among applicants.\n\nAnother study in the US looked at bias in the Research Project (R01) grants from the National Institutes of Health (NIH), and found this grant program exhibited gender bias in Type 2 renewal applications (Kaatz et al., 2016). The authors analysed 739 critiques of both funded and unfunded applications, using text analysis and regression models. The study found that reviewers gave worse scores to female applicants even though they used standout adjectives in more of their critiques. A second piece of work from the same authors employed more state-of-the art text mining algorithms to discover linguistic patterns in the critiques (Malikireddy et al., 2017). The algorithms showed that male investigators were described in terms of leadership and personal achievement while females were described in terms of their working environments and ‘expertise’—potentially suggesting an implicit bias where reviewers more easily view males as scientific leaders, which is a criterion of several grant funding programs.\n\nIn a longitudinal study, researchers followed the careers of an elite cohort of PhDs who started postdoctoral fellowships between 1992 and 1994 (Levitt, 2010). The study found that 16 years after the fellowships, although 9 per cent of males had stopped working in a scientific field, compared with 28 per cent of females, there was no significant difference in the fractions obtaining associate or full professorships. However, females whose mentors had an h-index in the top quartile were almost three times more likely to receive grant funding – males’ success had no such correlation with their mentors’ publication record.\n\nIn a Canadian Institutes of Health Research (CIHR) funded study, researchers evaluated all grant applications submitted to CIHR in the years 2012–2014 (Tamblyn et al., 2018). Descriptive statistics were used to summarise grant applications, along with applicant and reviewer characteristics. The dataset was then interrogated with a range of statistical approaches (2-tailed F-test, Wald χ2 test), which showed that higher scores were associated with having previously obtained funding and the applicant’s h-index and lower scores with applicants who were female or working in the applied sciences.\n\nSome funding agencies do not detect gender bias in their grant programs. For example, the Austrian Science Fund performed an analysis of 8,496 research proposals from the years 1999–2009 using a multilevel regression model, and found no statistically significant association between application outcome and gender (Mutz et al., 2012). Meta-analyses of gender bias have reported on both sides of the debate, with claims that applicant gender has little (Ceci & Williams, 2011) or substantial (Bornmann et al., 2007) effect on receiving grants.\n\nExploration in relation to racial bias has also been performed, though there is a smaller body of work than on gender bias. In 2011 researchers funded by the NIH showed that black applicants were ten percentage points less likely to obtain R01 funding than their white peers, after extensively controlling for external factors (educational background, country of origin, training, previous research awards, publication record and employer characteristics) (Ginther et al., 2011). A funding gap between white/mixed-race applications and minority applicants has been a persistent feature of NIH grant funding between 1985 and 2013 (Check Hayden, 2015). According to a preprint article from mid-2018, racial bias in the NIH system may have diminished (Forscher et al., 2018). The researchers report on an experiment where 48 NIH R01 proposals were modified to contain white male, white female, black male and black female names before being sent for review by 412 scientists. The authors found no evidence—at the level of ‘pragmatic importance’—of white male names receiving better evaluations than any other group; however, they note there may be bias present at other stages of the granting process.\n\nCareer stage. Career stage is another potential source of bias in the peer review process. An ageing population of researchers may cause problems because it may crowd-out early-career researchers from funding, thus preventing them from establishing their careers (Blau & Weinberg, 2017). In the US, the average career age of new NIH grantees, defined as years elapsed since award of the highest doctoral degree, increased dramatically between 1965 and 2005, rising from 7.25 years to 12.8 years (Azoulay et al., 2013). The cause of the shift is uncertain. Proposed theories include an increased burden of knowledge due to an expanding scientific frontier; the use of post-doctoral positions as a ‘holding tank’ for cheap, skilled labour; and the move to awarding grants as prizes for substantial preliminary results rather than to fund new research (Azoulay et al., 2013).\n\nMeasuring bias regarding career stage is problematic due to the challenges associated with defining career stage. Career age—the years elapsed since award of the highest doctoral degree—is one commonly used description (Azoulay et al., 2013). While this approach is suitable for identifying strong trends, such as the near doubling of career age of new NIH grantees discussed above, it does not take into account factors such as teaching commitments, changing research topics, clinical work or career breaks (Spaan, 2010).\n\nThere are other approaches to defining career stage, for example focusing on necessary competences rather than time elapsed. The European Framework for Research Careers has four stages—first stage researcher, recognised researcher, established researcher and leading researcher—and provides a classification system that is independent of career path or sector (EC-DGRI, 2011).\n\nResearch field. There may be biases between research fields and also against research that falls between, or combines, those fields. While interdisciplinary research is often considered fertile ground for innovation, there is a belief among researchers that interdisciplinary proposals are less likely to receive funding (Bromham et al., 2016). Defining and identifying interdisciplinary research is a challenge that has hindered the evaluation of this potentially damaging belief. A recent study sought to address this challenge by developing a biodiversity metric, the interdisciplinary distance (IDD) metric, to capture the relative representation of different research fields and the distance between them (Bromham et al., 2016). Using data from 18,476 proposals submitted to the Australian Research Council’s Discovery Program over a five-year period, the authors found that the greater the degree of interdisciplinarity, the lower the probability of an application being funded.\n\nInstitution. There is also some evidence that characteristics of the institution may be a source of bias in the grant application process. For example, a 2016 study of Canada’s Natural Sciences and Engineering Research Council (NSERC) Discovery Grant program found that funding success and quantity were consistently lower for applicants from small institutions, and that this finding persisted across all levels of applicant experience as well as three different scoring criteria (Murray et al., 2016). The authors analysed 13,526 proposal review scores, using logistical regression to determine patterns of funding success and developing a forecasting model that was parameterised using the dataset. The authors note that some differences between institutions may be due to differences in merit and differences in research environments; they recommend that more needs to be done to ensure funds are distributed appropriately and without bias.\n\nReviewers. Reviewers may have overt or implicit biases that can affect their scoring of grant proposals, some of which are noted above. The level of expertise that reviewers have relating to an application can affect their evaluations, with studies finding both advantageous and disadvantageous effects. Li examined this issue by constructing and analysing a dataset of almost 100,000 applications evaluated in over 2,000 meetings (Li, 2017). The study found an applicant was 2.2 per cent more likely to receive funding, the equivalent of one-quarter of the standard deviation, if evaluated by an intellectually closer reviewer as measured by the number of permanent reviewers who had cited the applicant’s work in the five years prior to the meeting. Conversely, another study found that reviewers with more expertise in an applicant’s field, as measured by a self-assessment of their level of expertise relating to an application, were harsher in their evaluations (Gallo et al., 2016).\n\nThe characteristics of reviewers have also been shown to affect their evaluations. Jayasinghe, Marsh and Bond have published several studies based on collaboration with the Australian Research Council (Marsh et al., 2008) exploring the peer review of grant applications. One finding was that the nationality of peer reviewers affected the ratings they gave, with Australian reviewers scoring similarly to European reviewers, but harsher than those from other countries and specifically North America. The authors were unable to determine if the cause was awareness that Australian reviewers were likely competing with applicants for funding, or purely a result of different nationalities.\n\nEven in the absence of bias, reviewers may not always agree on the quality of a proposal. The concept of inter-rater reliability—the degree of agreement among raters—is central to peer-review, yet has not been thoroughly examined in this context (Clarke et al., 2016). Three studies over the last half century have shown quite consistent levels of disagreement between reviewers, ranging from 24–35 per cent disagreement (Cole et al., 1981; Fogelholm et al., 2012; Hodgson, 1997). A more recent randomised trial study considered 60 applications to NHMRC’s Early Career Fellowship program, which were duplicated by NHMRC secretariat and reviewed by two grant panels (Clarke et al., 2016). The study found inter-rater reliability to be 83 per cent, which is comparable to the previous studies. The authors suggest that the slight reduction in disagreement may be due to the nature of early career applications or differences in the scoring and assessment criteria.\n\nAs the research community has gained an increasing awareness of bias, steps have been taken to develop fairer processes and programs. Table 5 below provides a summary of approaches used by a range of international funders both to reduce bias in their funding processes, and to evaluate bias across their funding streams.\n\nMany funders now have targeted grant streams to support applicants who were found to be disadvantaged by biases in peer review or program structure, such as early career researchers. For example, the NIH K02 Independent Research Scientist Development Award, the Medical Research Council (MRC) New Investigator Research Grant, and the European Research Council (ERC) Starting Grants, are a small selection of funding aimed at early career researchers.\n\nThere is some emerging evidence that training can reduce bias and increase the inter-rater reliability of reviewers. The CIHR introduced a reviewer training program following the discovery that its new grant system focusing on applicants’ track records was disadvantaging women, while a program focusing on the research proposal was not. In the grant cycle following the introduction of a training module on unconscious biases, female and male scientists had equal success rates (Guglielmi, 2018). Additionally, an online training video was found to increase the inter-rater reliability for both novice and experienced NIH reviewers, with correlation scores rising from 0.61 to 0.89 following training (Sattler et al., 2015).\n\nBlinding the identity of applicants from reviewers has been studied as a mechanism for increasing the fairness of peer review systems. In the context of journal peer review, the journal Behavioural Ecology found that its introduction of double-blind review increased the representation of female authors by 33 per cent, to reach a level that reflects the composition of the life sciences academic workforce (Budden et al., 2008). The US National Science Foundation (NSF) has trialled a blinded application process called ‘The Big Pitch’, which involves applicants submitting an anonymised two-page research proposal alongside a full conventional proposal (Bhattacharjee, 2012). The NSF reported that there was only ‘a weak correlation’ between the success outcomes of the full and the brief, anonymous applications.\n\nThe number of grant applications for research submitted for review is increasing in the majority of countries and disciplines (De Vrieze, 2017). However, funding for research is being reduced, leading to a decrease in the success rate of applications. The grant application process is time-consuming and costly, with the burden falling on those applying for the funding (Bolli, 2014; Gurwitz et al., 2014; Guthrie et al., 2018; Kulage et al., 2015), those reviewing the applications submitted (Bolli, 2014; Kitt et al., 2009; Schroter et al., 2010; Snell, 2015), the funding agency (Liaw et al., 2017; Schroter et al., 2010; Snell, 2015) and the research institutions (Kulage et al., 2015; Ledford, 2014; Specht, 2013).\n\nMeasuring burden in the funding process. The burden of the grant application process has been measured for applicants, reviewers, funders and research institutions using a variety of methods. A list of the different approaches used to evaluate burden of the application process can be found in Table 6.\n\nSurveys have frequently been used to assess the burden of grant preparation (Herbert et al., 2013; Herbert et al., 2014; Kulage et al., 2015; Wadman, 2010) and grant reviewing (Schroter et al., 2010; Wadman, 2010). These surveys enquire on the time spent on average in the application and review process, as well as the distribution of time among the various activities of the grant application process.\n\nAnother approach has been to use an observational study of proposals for health services research funding to measure time spent preparing proposals, the use of simplified scoring of grant proposals (reject, revise or invite to interview), and progression from submission to outcome (Barnett et al., 2015).\n\nOthers have estimated the cost of grant application by tracking the time spent preparing a proposal and combining the data with nationwide salaries, fringe rates, and facilities and administrative (F&A) cost recovery rates for personnel (Kulage et al., 2015).\n\nThe minimum number of reviewers has also been assessed in order to reduce the burden on reviewers. (Liaw et al., 2017; Snell, 2015). One study focused on NHMRC and looked at the agreement on funding decisions on applications between different numbers of panellists and different lengths of applications (Liaw et al., 2017). A second study evaluated the review process from a CIHR post-doctoral fellowship competition, bootstrapping replicates of scores from different reviewers to determine the minimum number of reviewers required to obtain reliable and consistent scores (Snell, 2015).\n\nIn recent years, different strategies have been developed to try to reduce the burden of grant applications. Table 7 provides a summary of approaches used by a range of international funders, both to reduce burden in their funding processes, and to measure the level of burden. These measures could allow researchers to focus on their research, save reviewers time, and potentially reduce the cost of grant review by reducing the labour required to review grant applications (Bolli, 2014).\n\n2 AQuAS is not a funding agency. AQuAs is a non-profit public assessment agency of the Catalan Ministry of Health (Spain).\n\nApplication limits. In 2009, the NIH incorporated a clause into their grant policy limiting applicants to two submissions of a research proposal (Rockey, 2012). Although this policy was not well received by the research community (Benezra, 2013), analysis by the NIH revealed a higher proportion of first-time applications were being awarded, along with a reduction in the average time to award from submission (Rockey, 2012). Restricting resubmission has also been adopted by the European Research Council (ERC) (Council, 2017).\n\nTwo-stage application process. The ERC has combined the application limit with a two-stage application that involves awarding project proposals a score during the first stage of the application process (A, B or C). If the project is awarded an A, the proposal will proceed onto the next assessment stage. If the proposal received a B, the applicant must wait one year before reapplying. And if the proposal is graded with a C, the applicant must wait two years before reapplying to any of the ERC-funded programs. This approach has led to a decrease in the number of applications received for evaluation by the ERC (INT04). The NIHR have also adopted a two-stage application process that has led to a decrease in the number of applications sent for peer review and a shorter time between application submission and outcome notification (INT01).\n\nMultiple calls per year. Moreover, the NIHR has multiple calls for proposals throughout the year, which reduces not only the burden on reviewers by decreasing the number of applicants to review per round (INT01), but also on the applicants by having ongoing grant applications (Herbert et al., 2014).\n\nGrant application length. In 2012, the NIH reduced the length of most grant applications from 25 pages to 12 pages, with the aim of reducing the administrative burden on both applicants and reviewers, and to focus on the concept and impact of the proposed research rather than the details of the methodological approach (Wadman, 2010). However, a study by Barnet et al. found that shortening the length of an application slightly increased the time spent by applicants preparing the proposal (Barnett et al., 2015).\n\nFunding period. Extending the funding period to five years (Bolli, 2014) has also been suggested to reduce the burden of grant application.\n\nWhat constitutes ‘innovative research’ is difficult to define. Approaches to identifying and defining innovation are varied and include defining innovation based on expert opinion (Marks, 2011), as research that does not require extensive preliminary results (Spier, 2002) (INT04), and (in the field of clinical research) as efforts that lead to improvements in patient care and progress (Benda & Engels, 2011).\n\nAlthough there is a belief that quality research should be innovative and lead to new understanding of science (Benda & Engels, 2011), the current process of reviewing grant applications, mainly peer review, has been defined as ‘anti-innovation’, providing strong incentives for incremental research and discouraging research into new unexplored approaches (Azoulay et al., 2013; Fang & Casadevall, 2016; Guthrie et al., 2018).\n\nInnovation and productivity are driven by diversity (Magua et al., 2017); therefore, advancing women or other underrepresented groups to institutional leadership (Magua et al., 2017) as well as promoting interdisciplinary research (Bromham et al., 2016) should have a positive impact on promoting innovative research.\n\nAnother feature of innovative research is its uncertain and potentially controversial nature. While many funding agencies aim to support innovative research, the body of work on peer review suggests it is an inherently conservative process (Langfeldt & Kyvik, 2010).\n\nSince defining and identifying innovative, creative research is challenging, it can be difficult to measure the level of innovation and creativity within a research portfolio, or the extent to which a research funding program fosters innovation. However, there are examples in the literature of potential approaches to measuring innovation, as set out in Table 8.\n\nBy definition, new ideas are likely not to be met with consensus. It has been suggested that innovation could be measured through a metric based on lack of agreement between reviewers, measuring controversy as a surrogate for innovation, with new metrics, including variance or negative kurtosis, the degree to which observations occur in the tails of the grading distribution (Kaplan, 2007).\n\nProductivity is another potential approach to measuring innovation. One study assessed the careers of researchers funded by two distinct mechanisms, investigator-initiated R01 grants from the NIH and the investigator program from the Howard Hughes Medical Institute (HHMI), with the aim of determining whether HHMI-style incentives result in higher rate of production of valuable ideas (Azoulay et al., 2011). The authors estimated the effect of the program by comparing the outputs of HHMI‐funded scientists with that of the NIH-funded scientists within the same area of research, who received prestigious early career awards. Using a combination of propensity-score weighting and difference-in-differences estimation strategies, the authors found that HHMI investigators produced more high-impact journal articles than the NIH-funded researchers, and that their research was more prone to changes.\n\nAnother study looked at the relation between the knowledge contained in an application proposal and a reviewer’s expertise and the outcome of proposals focusing on innovative research and area of expertise (Boudreau et al., 2016). In this study, the authors designed and executed a grant proposal process for research, and randomised how proposals and reviewers were assigned, generating 2,130 evaluator-proposal pairs. The authors found that evaluators give lower scores to research proposals that are highly novel, evaluated as new combinations of MeSH terms in a proposal relative to MeSH terms in the existing scientific literature, and to proposals in their area of expertise (Boudreau et al., 2016). However, another study focusing on public health proposals found that reviewers favour their own fields (Gerhardus et al., 2016).\n\nA few organisations conduct peer review, with some unique practices placing higher value on innovation (Liaw et al., 2017). Criteria for assessing innovation are determined by the different organisations.\n\nIn recent years, different strategies have been developed to improve the assessment of innovation in grant review. Table 9 provides a summary of approaches used by a range of international funders, both to increase innovation and creativity and to evaluate the level of innovation and creativity across their funding streams.\n\n3 AQuAS is not a funding agency. AQuAs is a non-profit public assessment agency of the Catalan Ministry of Health (Spain).\n\nTo ensure innovative research is being funded some agencies, including the NIH, adopt an ‘out of order funding’ approach (Lindner & Nakamura, 2015). In this approach, a number of applications for innovative research are chosen for funding despite receiving lower scores than other funded research based purely on the peer review process. In the NIH, this strategy has led to approximately 15 per cent of applications selected ‘out of order’.\n\nThe NIH has also made additional changes to the peer review process in order to increase the emphasis on innovation and decrease the focus on methodological detail (Lindner et al., 2016). These changes included reducing the length of the methodological description (from 25 to 12 pages), with guidance to focus away from routine methodological details towards describing how their application is innovative. Including innovation as a criterion for grant assessment could incentivise researchers to include innovative ideas and new approaches into their proposals (Guthrie et al., 2018).\n\nMany funding agencies have also adopted the strategy of having a separate scheme to fund innovative research, allocating smaller funds with a shorter time frame to these specific streams. The NIH has developed the New Innovator Award, committing $80 million to the award, and two others that specifically encourage innovation, the Pioneer and Transformative R01 Awards (Alberts, 2009). ZonMW have designed an ‘off-road’ program aimed at high-risk, high-reward projects, providing €100,000 for 1.5 years (INT02). NIHR has also designed different funding tiers to promote funding for innovative projects, providing £150,000 funding for 18 months (INT01). However, this strategy could include longer funding periods to encourage a culture of innovation among young researchers who remain reluctant to take the risk of pursuing ambitious ideas, acknowledging the need for preliminary results to obtain funding for most research (Alberts, 2009).\n\n\nDiscussion\n\nOur review of international practice regarding the characterisation and measurement of bias, burden, and conservatism innovation and creativity in the grant funding process demonstrated that the efforts so far systematically to measure these characteristics by funders have been limited. However, in each area there were examples of existing practice we can draw upon as summarised in Table 10.\n\nIt is also worth noting the challenges in defining each of these elements, partly reflecting the diversity within each of these areas. In terms of bias, we note biases can emerge in terms of a range of areas, with five main areas highlighted in the literature: applicant characteristics (e.g. gender, ethnicity); career stage; research field; institution; and reviewer characteristics. Burden can be characterised in terms of where the burden is experienced: by applicants, reviewers, the funding agency and by institutions. Efforts to address burden and ways of measuring their effectiveness may differ across these groups. Finally, a key challenge in measuring innovation is providing a definition of innovative or creative research that can be operationalised. Often funders do this based on expert judgement, but this is challenging to use for portfolio assessment and analysis.\n\nFinally, a key limitation of the work is that since this is a review of the existing literature and practice, we are constrained by what has so far been reported, which in some areas is fairly limited. In particular, the majority of the literature focuses on the application and peer review process, which only forms a part of the overall funding scheme that starts from the initial establishment of the structure of the funding scheme through to the monitoring and evaluation of ongoing and completed funding awards. We set out in Table 11 a wider conceptualisation of some of the ways in which challenges could theoretically emerge in relation to funding schemes at different stages throughout this process. This is intended to illustrate the potential breadth of scope for this work beyond the literature: as such it is neither exhaustive nor driven by existing evidence of those challenges or opportunities emerging in practice. Rather it acts as an aid to thinking through the full process of the development and implementation of funding schemes. We suggest that further research and evaluation efforts are needed to more fully conceptualise and measure effectively the concepts of bias, burden and innovation in research across the full scope of the research funding process.\n\n\nData availability\n\nFigshare: Articles on bias burden and conservatism in grant peer review. https://doi.org/10.6084/m9.figshare.8184113.v1 (Guthrie, 2019).\n\nThis project contains a list of all publications, with URLs, identified during the literature review.\n\nConsent sought and confidentiality assured in the interview process means that the interview transcripts from this study cannot be made publicly available (see interview protocol Table 3). This is due to the high risk of identifying individuals from the small sample of interviews conducted.", "appendix": "Grant information\n\nThis work was funded by the National Health and Medical Research Council (NHMRC) of Australia.\n\nThe funders had no role in the study design, data collection and analysis.\n\n\nReferences\n\nAlberts B: On incentives for innovation. Science. 2009; 326(5957): 1163. PubMed Abstract \| Publisher Full Text\n\nAzoulay P, Graff Zivin JS, Manso G: National institutes of health peer review: Challenges and avenues for reform. In: Innovation Policy and the Economy. 2013; 13: 1–21. Publisher Full Text\n\nAzoulay P, Zivin JS, Manso G: Incentives and creativity: evidence from the academic life sciences. Rand J Econ. 2011; 42(3): 527–554. Publisher Full Text\n\nBarnett AG, Herbert DL, Campbell M, et al.: Streamlined research funding using short proposals and accelerated peer review: an observational study. BMC Health Serv Res. 2015; 15(1): 55. 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PubMed Abstract \| Publisher Full Text\n\nSpier RE: Peer review and innovation. Sci Eng Ethics. 2002; 8(1): 99–108; discussion 109–112. PubMed Abstract \| Publisher Full Text\n\nTamblyn R, Girard N, Qian CJ, et al.: Assessment of potential bias in research grant peer review in Canada. CMAJ. 2018; 190(16): E489–E499. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomkins A, Zhang M, Heavlin WD: Reviewer bias in single- versus double-blind peer review. Proc Natl Acad Sci U S A. 2017; 114(48): 12708–12713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Lee R, Ellemers N, Fiske ST: Gender contributes to personal research funding success in The Netherlands. Proc Natl Acad Sci U S A. 2015; 112(40): 12349–12353. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWadman M: Shorter NIH grant form launches. Nature. 2010; 463(7277): 12–13. PubMed Abstract \| Publisher Full Text\n\nWennerås C, Wold A: Nepotism and sexism in peer-review. Nature. 1997; 387(6631): 341–3. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49804", "date": "18 Jun 2019", "name": "Adrian Barnett", "expertise": [ "Reviewer Expertise statistics", "meta-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 is important research given the impact of research funding on what science is funded and the flow-on benefits of research to the public. The authors examined the recent literature, but very usefully they also interview funders to find out what is happening in practice. I think this review could be very useful for funders and for researchers working in meta-research.\n\nMy main issue was that important detail was lacking in a number of places concerning the strategies and changes used by funders. This is a list of places where I wanted more detail, which are mostly based on text in the tables:\nTable 5, the German Research Foundation have 300 equality measures. Is this a typo? That many measures seems to be far too many to be useful, and the funders could just end up drowning in information. Table 5, the German Research Foundation evaluate bias using \"Progression of careers\" and \"Institutional bias\" but some idea of how they do this would be useful. Do they only examine gender bias? Do they take any action if they detect bias? Table 5, NIHR, do they provide more money for the higher tiers? Table 5, NIHR, they evaluate bias using \"Diversity of applications\", but in terms of what? Gender and race? Table 5, ARC, does the ROPE statement have any impact? Especially as some Australian researchers have said that ROPE is \"largely perceived as a tokenistic gesture put on forms and never taken into account by the people who make decisions and evaluate work\".1 Table 5, ARC, what does \"ongoing access to all research projects\" mean? Access for who? The ARC or the public? Table 5, ARC, they look at the discrepancy in scores, but what action do they take when they find a discrepancy? Table 5, ARC, is international benchmarking sensible given that funders in other countries could also be struggling with biases in terms of gender or race? Wouldn't it be better to set targets/benchmarks based on consultation with the research community? Table 7, German Research Foundation, how does have nine different schemes reduce burden? It could have the opposite effect if people apply for multiple schemes to increase their chance of winning some funding. Table 7, Medical Research Council, how does a report on the conduct and outcome of a project reduce burden? Table 7, ZonMw, does the pre-screening of applications based on CVs reduce burden because the reviewers therefore see fewer applications? Table 7, ZonMw, how does the mid- and end-point review evaluate burden? Table 9, CIHR, how does having over 50 percent of funding at investigator-driven research increase innovation? What if investigators only put forward conservative ideas? See for example work by Nicholson et al.2 Table 9, ARC, how does a continuous funding round increase innovation or creativity? I can see how this could increase diversity by being more accommodating of researchers with family commitments. Table 9, ERC, \"External program evaluation\" needs more detail Table 10, more detail is needed for \"Longitudinal\", does this mean following those who did and did not win funding? Table 11, I did not understand the row entries in the table concerning \"personal content of applications\"\n\nMinor comments\nIs some mention of the EVIR (Ensuring Value In Research) collaboration of funders merited? Page 8, more cautious language may be needed when using the Levitt study given that it was an exploratory study that does not mention a protocol. In particular I am concerned about the use of quartiles as a threshold, as it's not clear if other thresholds were tried (e.g., tertiles, quintiles). Page 10, in terms of the Clarke et al study - for which I was an author - we actually concluded that the inter-rater agreement appeared higher than in previous studies, so I would not say 'comparable'. I think the most likely reason for this greater agreement was that our study concerned people-funding where the main idea is to rank past performance, whereas all other previous studies had examined project-funding where the main task is to predict future performance which is far harder. Table 6, order the rows by date or first author name? Table 6, Schroter et al 2010 row, the first paragraph in the \"Methodology\" column end abruptly. Table 6, Barnett et al 2015 study (for which I am an author), the streamlined funding did not lead to an increase in success rates because all the rounds were streamlined. The increase in success rates over time occurred because the scheme had far fewer proposals over time. Many of the proposals in the initial rounds were ineligible because it was a new funding scheme and a lot of researchers simply \"had a go\". Page 16, first column, first paragraph, the NIH \"two-chances\" policy has since been relaxed, see work by Kaiser.3 Page 16, paragraph \"Grant application length\", it's fairer to say that a shorter form \"was associated with\" an increased application time, as this was a non-randomised study. Page 17, some funding schemes have used a \"wildcard\" option for panels, where each panel member was allowed to save one proposal from a culling stage. I cannot find the reference for this, but I can find where we have discussed this idea.4 There was little on the transparency of funders. If funders opened up the anonymised application data for scrutiny, then this could be a great way of assessing innovation and diversity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "49801", "date": "17 Jul 2019", "name": "Robyn Tamblyn", "expertise": [ "Reviewer Expertise Epidemiology", "Health Information Systems", "Health Services", "Medical Education", "Medical Informatics", "Pharmacoepidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the work clearly and accurately presented and does it cite the current literature?Partly...\nThe introduction fails to motivate the rationale for this study, specifically examining the level of burden, evidence of bias, and support for innovative and creative research in peer review for grant applications. This section was disappointing but some of this rationale is buried in the results section and should be re-organized.\n\nIs the study design appropriate and is the work technically sound? Partly...\nThe processes for selecting for reviewing the quality of the articles is not specified and there are many standards available. Research in relationship to research funding may not presented in a standard research format but there are many quality metrics that can be used to evaluate the research and these need to be incorporated.\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes…\nThe interview form is provided as part of the manuscript.\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly..\nIn reviewing the results from other studies, the way in which the statistical analysis are represented was rather odd. For example, the author talked about correlation for binary variables. This is inappropriate. The author also talked about the correlation between a short anonymized form and long form which would be a difficult way of looking at agreement on success rates which is also a binary variable. In referring to the CIHR study the author talked about detailed F tests and X2 tests without referring to the nature of the underlying multivariate analysis which seems strange as these are only tests of statistical significance and not the actually underlying process used to estimate the association. It would helpful to have a statistical reviewer as part of the review team for these articles to assist in the interpretation of the study results and the respective statistics.\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…\nJust in terms of commentary. The Results section was very informative and the tables summarizing different approaches was also very useful. It was difficult to understand the choice of funders to review and it was very surprising to see the NIH excluded from the interviews, although the research that has been done on NIH peer review are included in the results section. It is also strange to make the comment that findings from one funder are not generalizable to another. The literature shows that there are trends that cut across funding agencies, countries and even between manuscript and grant review and I think that this statement is not at all supported by the results that are presented.\n\nFinally, the section on innovation was very important and will be an addition to the literature. Results that were presented do not support the statement that funding agencies are not really doing much work in this area although I think that is likely true. Basically, the author outlined what the finding agencies were doing and it was left to the reader to interpret that some of these are very generic approaches that really do not address the key issues such as burden or bias.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-851
https://f1000research.com/articles/8-850/v1	12 Jun 19	{ "type": "Study Protocol", "title": "Rationale and design of perioperative myocardial ischemia: a protocol for troponin monitoring, prognostic thresholds, economic analysis and further insights into pathophysiology for non-cardiac surgery patients", "authors": [ "Ekaterine Popova", "Pilar Paniagua Iglesias", "Jesus Alvarez Garcia", "Miguel Vives Borras", "Francesc Carreras Costa", "Xavier García-Moll Marimón", "Mercedes Pilar Rivas Lasarte", "Aranzazu Gonzalez Osuna", "Cecilia Martinez Bru", "Adria Font Gual", "Ruben Diaz Jover", "Inmaculada India Aldana", "Gonzalo Azparren Cabezon", "Misericordia Carles Lavila", "Montserrat Rué Monné", "Javier Zamora Romero", "MªJosé Martinez Zapata", "Pablo Alonso-Coello", "Pilar Paniagua Iglesias", "Jesus Alvarez Garcia", "Miguel Vives Borras", "Francesc Carreras Costa", "Xavier García-Moll Marimón", "Mercedes Pilar Rivas Lasarte", "Aranzazu Gonzalez Osuna", "Cecilia Martinez Bru", "Adria Font Gual", "Ruben Diaz Jover", "Inmaculada India Aldana", "Gonzalo Azparren Cabezon", "Misericordia Carles Lavila", "Montserrat Rué Monné", "Javier Zamora Romero", "MªJosé Martinez Zapata", "Pablo Alonso-Coello" ], "abstract": "Introduction: Worldwide, near 200 million adults undergo major non cardiac surgery\n\neach year, and 10 million of them are estimated to suffer a myocardial injury after non-cardiac surgery (MINS), defined as an elevated high sensitive troponin T (hs-cTnT) in the first 3 days after surgery. Troponin levels need to be monitored in order to diagnose MINS, high sensitive cardiac Troponin T (hs-cTnT) assays being currently the most frequently used. Perioperative hs-cTnT screening could lead to care decisions that can potentially improve clinical outcomes. However, many of the clinical and economic implications of perioperative hs-cTnT monitoring remain unclear, and need to be elucidated. Methods and analysis: Prospective cohort that will include patients with high cardiovascular risk undergoing major non-cardiac surgery, expected to require at least an overnight hospital admission. Three determinations of hs-cTnT in each patient (before surgery, at 48, and 72 hours after surgery) will be obtained. We will determine the incidence and prognosis of MINS, and calculate prognostically relevant thresholds for pre- and post-operative hs-cTnT. We will also conduct a cost-effectiveness analysis of hs-cTnT screening, compared with usual care. Finally, using computed tomography angiography (CTA) and cardiac magnetic resonance imaging (MRI), we aim to elucidate further the pathophysiology of MINS. Ethics and dissemination: Our center had Ethics approval before including patients. Written informed consent is required for all patients before inclusion. The study will evaluate the feasibility and impact of implementing an hs-cTnT monitoring program at a tertiary hospital, as well as its cost-effectiveness, determine pre and postoperative thresholds of hs-cTnT and finally, evaluate potential mechanisms involved in perioperative ischemic events. The dissemination plan includes publishing the results in a policy-influencing journal, conference presentations, engagement of influential medical organizations, and taking published results to real practice.", "keywords": [ "Myocardial Ischemia", "PMI", "MINS", "hs-cTnT", "cost-effectiveness", "CT-angiography", "MRI." ], "content": "Strengths and limitations of this study\n\nOur study will evaluate the feasibility and impact of implementing a high sensitive cardiac Troponin T (hs-cTnT) monitoring program in patients undergoing non-cardiac surgery, and will inform preoperative and post-operative prognostically relevant thresholds.\n\nThe study will also determine the cost-effectiveness of hs-cTnT monitoring compared with usual care.\n\nOur cardiac imaging sub-study is the first case-control cohort application of non-invasive advance imaging diagnostic tools, computed tomography angiography (CTA), and cardiac magnetic resonance imaging (MRI) with the objective to better understand the pathophysiology of myocardial injury after non-cardiac surgery (MINS).\n\nDuring the implementation of hs-cTnT monitoring, some troponin measurements and, in consequence, some of MINS events may be missed in patients who do not experience ischemic symptoms.\n\nDue to the case-control design of the cardiac imaging sub-study, there might be difficulties to include healthy controls, as they might be reluctant to undergo further diagnostic testing.\n\n\nIntroduction\n\nWorldwide, annually over 200 million adults undergo major non-cardiac surgery1 and this number is growing continuously. Despite preoperative screening, technical improvement and early detection during clinical screening, perioperative myocardial injury (PMI) remains the first cause of morbidity and mortality within 30 days of surgery2. Available evidence indicates that patients undergoing non-cardiac surgery with only elevated cardiac markers reflecting cardiac injury, such as troponin, have a very poor prognosis3. However, most of these patients do not experience ischemic symptoms, and do not fulfill criteria for conventional clinical diagnosis of myocardial infarction (MI).\n\nThe clinical profile and short-term prognosis of patients undergoing non-cardiac surgery, was described in one of the largest multicentre international cohorts (VISION study)4, suggesting a new entity called myocardial injury after non-cardiac surgery (MINS)5, defined as troponin T elevation (≥0.03 ng/ml) in the first 3 days post-surgery. The definition of MINS is broader than the definition of MI as it also includes other prognostically relevant PMIs due to ischemia, but does not include PMIs due to non-ischemic causes (e.g. pulmonary embolism, sepsis, or cardioversion)5. Another recent prospective diagnostic study confirmed that despite early detection during routine clinical screening, PMI is associated with substantial short- and long-term mortality6. Therefore MINS, usually undetected by the absence of ischemic symptoms, is the most common major cardiovascular complication after non-cardiac surgery7, with more than 10 million patients potentially suffering these complications annually worldwide4–7.\n\nTroponin is the only available biomarker which helps to identify and manage MINS patients, providing rapid, specific and sensitive detection4–7. Routine monitoring for perioperative cardiac biomarkers, with the most frequently used high-sensitive cardiac Troponin T (hs-cTnT) assay, leads to recognize most of MINS and may improve prognosis. Preoperative hs-cTnT concentrations are also associated with postoperative MI and long-term mortality after non-cardiac surgery8. Typically, hs-cTnT monitoring is determined only in the post-operative period4, and despite the fact that some studies have determined hs-cTnT within 30 days before surgery6, a preoperative threshold has not yet been established.\n\nIn order to prevent missing this prognostically relevant event, nowadays guidelines recommend monitoring perioperative troponin in high-risk patients undergoing major non-cardiac surgery9,10. However, little is known about the economic consequences of troponin monitoring11, therefore economic evaluations of troponin monitoring compared to usual care, are needed. The implementation of postoperative troponin monitoring seems cost-effectively, particularly in patients at high risk for MINS12.\n\nFinally, despite a lot of recent interest mechanisms underlying MINS remain unclear13. There is laboratory, autopsy, imaging, and clinical evidence suggesting that coronary artery thrombosis may be one of the main pathophysiological mechanisms14,15. Theoretically, myocardial injury may originate from four main distinct mechanisms: coronary plaque rupture13,14, a myocardial oxygen supply-demand mismatch15,16, non-ischemic cardiac disorders such an atrial fibrillation episode17, or a non-cardiac cause as pulmonary embolism18. Minimally invasive diagnostic tools such as computed tomography angiography (CTA), together with cardiac magnetic resonance imaging (MRI), could help understand underlying mechanisms of MINS, and potentially improve the management and prognosis of these patients.\n\nGiven the above, we will evaluate the feasibility and impact of the implementation of routine hs-cTnT monitoring for the detection of prognostically relevant myocardial injury, determine hs-cTnT thresholds that could best predict short and long-term prognosis, conduct a full cost-effectiveness analysis, and further elucidate the pathophysiological mechanisms of MINS in high-risk patients undergoing major non-cardiac surgery.\n\n\nMethods\n\nThe study is divided in three sub-studies.\n\n\n1) Hs-cTnT screening programme implementation and clinical evaluation\n\nTo implement a hs-cTnT monitoring program in high-risk non-cardiac surgical patients\n\nTo determine the incidence and prognosis of MINS detected by a monitoring program\n\nTo determine which cut-off points of hs-cTnT better discriminate patients with death and/or MACE (myocardial infarction, unstable angina, congestive heart failure, new atrial fibrillation, stroke or pulmonary embolism) events (at 30 days and at 1 year) from those without.\n\nProspective cohort.\n\nWe will include adults ≥65 years or <65 years with documented cardiovascular disease (history of coronary artery disease, chronic heart failure, stroke and peripheral vascular disease), undergoing a major non-cardiac surgery (intraperitoneal, intrathoracic, major vascular, major orthopaedic, emergency) and expected to require at least an overnight hospital admission, that meets inclusion criteria, and no exclusion criteria (Table 1).\n\nHistory of coronary artery disease, chronic heart failure, stroke, or peripheral vascular disease.\n\nResearch personal will screen all surgical patients daily, both scheduled and emergency, to identify eligible patients. Potentially eligible patients will be approached to obtain informed consent after surgery and before hospital discharge. Template informed consent forms to be used are available as Extended data19.\n\nWe will measure hs-cTnT using a Roche Cobas e601 analyser (limit of detection 5.0 ng/L, 99th percentile 14 ng/L, 10% coefficient of variation at 13 ng/L) at three predefined time points: preoperatively (during the preoperative visit or just before surgery), and at 48 h and 72 h after surgery. If a rise and/or fall of hs-cTnT with at least one value above the 99th percentile upper reference (14 ng/L) is detected, a cardiologist will perform a clinical evaluation for possible MINS related symptoms, and a 12-lead electrocardiogram (ECG). If the post-surgery ECG has no changes, compared with ECG before surgery, we will conduct an echocardiography. In all included patients the cardiologist will conduct a structured clinical evaluation that will include the revision of current relevant medication (ASA and other antiplatelet, ACE inhibitors, statins, beta-blockers, anticoagulants). Cardiologists will discuss all treatment decisions derived from this visit and will discuss with treating surgeons (see Figure 1).\n\nA Coordination Committee will carry out periodic meetings to develop and assess the optimal implementation of the hs-cTnT monitoring strategy. Throughout the study we will have periodic meetings with, surgeons, anaesthesiologists, cardiologists, internists, and biochemistry personnel to explore barriers and perceptions about the monitoring program implementation. Alternatively, in case of detecting difficulties the Committee will propose potential solutions or alternative strategies. The Committee will include surgeons from the main surgical departments (orthopaedic, vascular, general, thoracic, plastic, otorhinolaryngology, and neurosurgery), anaesthesiologists, internists, and clinical epidemiologists.\n\nTo improve compliance with the screening program, we will aim to implement electronic solutions as much as possible, including the adaptation of electronic preoperative requests templates. For post-operative hs-cTnT measurements at 48 h and 72 h after surgery, we will involve corresponding treating surgical departments, and aim to implement automatically alerts on the electronic health records, as well as for cardiologist consultations. While these strategies are implemented, study personal will guarantee the optimal compliance of all circuits during the study period. Gradually the goal is that the monitoring program is run without the need of additional study personal, and that it is embedded within clinical routine.\n\nWe will follow-up all recruited patients during hospitalization, at 1 month and at 1 year after the date of surgery. The 1-month and 1-year follow-up visits will be performed by telephone. If the patients (or relatives) indicate that they have experienced any of the main outcomes, we will obtain the relevant source documents (Table 2).\n\nHigh-sensitive cardiac Troponin T before surgery and at 48 and 72 hours after surgery.\n\n**ASA and other antiplatelet, ACE inhibitors, statins, beta-blockers, and anticoagulants.\n\nWe estimated the sample size considering our experience with previous perioperative studies in our hospital (Hospital de la Santa Creu i Sant Pau) and a pilot study. In the hospital, approximately 80–100 patients per month (up to 1,000 patients per year) undergo major non-cardiac surgery. We therefore expect to recruit approximately 60–65 patients per month and a total of approximately 1,500 patients over a two-year period. From an estimated incidence of death (1–2%)4,7, and of major adverse cardiac events (MACE) (myocardial infarction, unstable angina, congestive heart failure, new atrial fibrillation, stroke or pulmonary embolism) (8–10%), we expect to observe 15–30 deaths and approximately 120–150 MACE composite events. We also expect to observe a 10% incidence of MINS (150 cases). This sample size will allow as estimating incidence of MINS, death and MACE events with a precision greater than 1.5% for the corresponding 95% confidence intervals.\n\nWe will obtain all data about hospital management (baseline, operative, and hospital discharge), and after discharge at one month and one year. We will collect the number of hs-cTnT measurements ordered by clinicians. We will also register the number of performed structured cardiologist visits. We will register occurrence of MINS, MACE, death and changes of medication during hospitalization, at 1 month and 1 year after surgery. Study personnel will collect data on case report forms (CRFs) and enter this information in a secure computerized database. Patients will be identified using a unique numeric code, and all patient data will be anonymized to ensure confidentiality. We will conduct periodically (every quarter) data validity checks ensuring data quality.\n\nOur main dependent variables will be screening coverage, and the incidence of MINS, death and MACE (myocardial infarction, unstable angina, congestive heart failure, new atrial fibrillation, stroke or pulmonary embolism). The main independent variables will be: age, sex, type of surgery, troponin measurement, cardiac risk index, history of coronary artery disease cardiac arrest, congestive heart failure, peripheral vascular disease, stroke, transient ischemic attack, chronic renal failure, deep venous thrombosis or pulmonary embolism, diabetes, hypertension, current atrial fibrillation, and chronic obstructive pulmonary disease.\n\nWe will describe variables according to their nature. We will provide the percentage and the number of cases for categorical variables. For quantitative variables, we will provide the mean and standard deviation. In terms of inferential statistics (relationships between variables), for prevalence and/or incidence we will calculate the corresponding 95% confidence intervals. We will explore univariate associations of independent variables to main outcomes (death and MACE) using chi-square test or Fisher’s exact test, and t-tests or non-parametric tests as needed.\n\nWe will conduct multivariate analysis using a binary logistic regression model to explore which factors are associated to MACE. The variables entered into the multivariate model will be those that showed statistically significant association in the univariate approach, and those that are considered as clinically relevant. We will obtain a final risk model following a backward elimination strategy. We will assess goodness of fit using the Hosmer-Lemeshow test, and a coefficient of calibration using C statistic to estimate the area under the ROC curve.\n\nFinally, from receiver-operator characteristic (ROC) curve analysis, we will estimate the best pre and post hs-cTnT cut-off values to predict 30 day and 1 year after surgery mortality and MACE. We will select the cut-off that maximizes Youden's index, and select secondary cut off values to achieve sensitivities of 80, 85, 90, and 95%. All tests will use a 5% (alpha = 0.05) significance level and will be two-tailed. We will use SPSS V 25.0 for all the analysis.\n\n\n2) Cost-effectiveness analysis\n\nTo evaluate the cost-effectiveness of a hs-cTnT monitoring program for the detection of MINS, compared with current practice (no screening).\n\nWe will develop an economic model considering two alternatives: the application of an hs-cTnT monitoring program for the detection of MINS/MI versus current practice, based on the application of standard treatments in the presence of ischemic symptoms.\n\nOur model will include the elaboration of a decision tree for short-term analysis, with a follow-up of patients at 30 days, and a Markov model for long-term analysis (lifetime). Both analyses will be develop from the perspective of the Spanish National Health System (SNS). In the long-term analysis, both costs and effects will be discounted at an annual rate of 3%, as recommended by the economic evaluation guides, and annual Markov cycles will be used.\n\nIn terms of items of direct healthcare costs, valued in Euros 2018, we will consider the following:\n\nMonitoring costs: hs-cTnT tests costs, health professional’s fees, laboratory technician fees, and administrative costs of implementation of the hs-cTnT monitoring program.\n\nDiagnostic tests cost: health professionals (cardiologist, nurse) fees, and consumables materials.\n\nCosts of treatment: hospitalizations, outpatient visits, and other hospital costs\n\nFollow-up patients cost: administrative costs\n\nGiven the perspective of the study, we will not include the non-health care costs and indirect costs. The health effects will be expressed as quality-adjusted life years (QALYs) in the lifetime study, and by avoided events in short-term study. We will obtain patients’ utilities from local data20 and the available research literature, searching in MEDLINE, PubMed, and PMC in our literature search. We will use use the following search terms: perioperative medicine, high-sensitivity Troponin T monitoring, Perioperative Myicardial Ischemia, MINS, Troponin T cut-off point, Troponin T monitoring cost-efectivness, computed tomography angiography (CTA), and cardiac magnetic resonance imaging (MRI).\n\nWe will estimate the incremental cost-effectiveness ratio (ICER) and we will perform sensitivity analysis of the key parameters. We will conduct a probabilistic sensitivity analysis to develop an acceptability curve in the long-term. We will present the results of the study separately; especially by temporal perspective, patient’s age (eg ≥65 years vs. <65 years) and MINS risk group (e.g. MINS vs. MI).\n\n\n3) Cardiac imaging\n\nTo clarify the underlying mechanisms involved in MINS and PMI in high cardiovascular risk patients undergoing non-cardiac surgery.\n\nNested case-control study.\n\nPatients from sub-study 1 will also be included in this sub-study (see eligibility criteria in Table 1).\n\nLocal pilot data21 shows an incidence of MINS of 10%, and a prevalence of significant coronary atherosclerosis identified by CTA in asymptomatic cardiovascular high-risk Mediterranean people of 18.9%22. We will need 130 patients with MINS/MI and 130 matched controls (adjusting by sex, age within five years interval, type of surgery and Lee index), accepting an alpha risk of 0.05, a beta risk of 0.2 in a two-sided test, and a loss rate of patients of 10%. We will validate our assumptions for this calculation once half of the sample is recruited.\n\nWithin the postoperative hospitalization period, we will approach eligible patients with the specific sub-study informed consent for the CTA and MRI exams. We will distinguish two groups of patients: cases and controls (Table 3).\n\nWe will perform CTA after 30 days of hospital discharge at Hospital de Sant Pau (Barcelona). Few days before the CTA, a cardiologist will treat patients with a beta-blocker (atenolol 25–50 mg or ivabradine 5–7.5 mg to achieve a heart rate of approximately ≤60 beats per minute). Expert evaluators (cardiologist or radiologist with level 3 training in interpretation of coronary computed tomography angiography), will read each angiogram using a 17-segment model of the coronary arteries, without knowledge of the clinical data. Each scan will be scored as normal (no evidence of coronary atherosclerosis); non-obstructive coronary artery disease (evidence of at least one coronary artery plaque with a <50% stenosis); obstructive coronary artery disease (at least one coronary artery plaque with a ≥50% stenosis); or extensive obstructive disease (≥50% stenosis in two coronary arteries including the proximal left anterior descending artery, ≥50% stenosis in three coronary arteries, or ≥50% stenosis in the left main coronary artery).\n\nAfter each CTA, we will conduct a MRI exam for every patient to analyse the global and segmental cardiac contractility, an assessment of necrosis and myocardial viability by studying late gadolinium enhancement contrast, and a pharmacological stress test with adenosine in the cases of CTA with obstructive coronary artery disease. After a matching analyses for the principal confounding factors (age, sex, Lee index, and type of surgery), one control for each case will be selected to complete the same CTA and MRI studies.\n\nWith the available findings, after the two tests, complemented with clinical anamnesis and relevant ECG changes, MINS patients will be classified into one of the following categories:\n\n• Plaque rupture\n\n• Supply-demand mismatch\n\n• Non-ischemic cardiac cause\n\n• Non-cardiac cause\n\nWe will collect all perioperative clinical data. We will include the following variables: demographic (age and gender), therapeutic (previous medical treatment with aspirin, other antiplatelet agents, statins or beta-blockers), related diagnostic tests (sensitivity), risk factors (Lee index and type of surgery), comorbidities, and perioperative data.\n\nWe will describe variables according to their nature. We will obtain absolute and relative frequencies for categorical variables. We will provide the mean and standard deviation for quantitative variables. We will estimate the prevalence of each one of the proposed aetiologies along with their exact 95% confidence intervals in the MINS sub-cohort, and compare these prevalence to the ones observed in the non-MINS cohort. We will assess if there is an association between aetiology and MINS status using contingency tables of each aetiology in the two groups (MINS and non-MINS), along with chi-square test or Fisher’s exact tests for the evaluation of the statistical association. In case of any clearly distinct covariate disbalance, a multivariate approach will be used. We will assess the agreement between diagnostic findings in the MINS population and control group using Cohen’s kappa coefficient. For the significance level we will use a 5% (alpha = 0.05) bilateral approach. We will use SPSS V 25.0 for all the analysis.\n\nThe study is coordinated by the Hospital de la Santa Creu i Sant Pau (Barcelona), which is primarily responsible for the organization of the study, development, study database, ensuring data quality, ensuring monitoring, coordination of the sub-studies, and data analyses. This study is part of a research line of perioperative medicine, which explores strategies for diagnosis, prevention, treatment, and risk prediction that promotes better management of patients undergoing surgery. The research team of this study is a multidisciplinary group that combines clinical investigators from epidemiology, anesthesiology, cardiology and biochemical departments, with large experience in the perioperative medicine area. The study structure includes an Adjudication Committee, an Operations Committee, and Steering Committee. The Adjudication Committee composed of clinicians who have expertise in perioperative outcomes, adjudicates all important clinical events. The Steering Committee supervises the whole project, and Operations Committee is in charge of the day to running of the study. Each sub-study has one lead investigator, and the project has one principal investigator (PAC). Given that it is an observational cohort it was considered not necessary to have a monitoring committee.\n\nThe protocol has received approval and consent from the Ethics Committee of Clinical Research Institute of our centre. Research personnel or good clinical practice trained health care professionals will obtain written informed consent for each patient. All data will be stored on a central encrypted, high-security computer system and kept strictly confidential.\n\nIn the case-control sub-study that includes CTA and MRI explorations, we will be contracted a specific insurance for controls. In case of patients with MINS, the CTA and MRI tests are clinically justified.\n\nThe knowledge dissemination plan includes traditional modes of dissemination (i.e., publication in a policy-driving journal, national/international conference presentations), as well as engagement of influential medical organizations. Broader dissemination will be performed by the Biomedical Research Institute Sant Pau (IIB Sant Pau) public website, and Twitter account. Also dissemination will be conducted in the Spanish Association of Anesthesia and Cardiology, as well as in our international network of perioperative medicine\n\nApproval and consent was received from the Ethics Committee of Clinical Research Institute of the Hospital de la Santa Creu i Sant Pau, for Protocol version: 1.1. Date: 2016-05-09. The study is currently in progress, having screened 1,900 patients, and recruited a total of 1,200 patients. Patient recruitment was initiated in 2016 and will end in 2019.\n\n\nDiscussion\n\nWe will evaluate feasibility and impact of the implementation of routine hs-cTnT monitoring for the detection of prognostically relevant myocardial injury. Our proposal will also aim to determine the hs-cTnT threshold that could best predict short and long-term prognosis. Given the scarce evidence regarding the economic aspects of troponin monitoring our cost-effectiveness analysis will provide new important knowledge in this area. Finally, by the application of the advance imaging techniques (CTA and MRI), this proposal will provide further insights in the identification of the mechanisms of MINS in high-risk patients undergoing major non-cardiac surgery.\n\nTroponin monitoring. Available evidence indicates that among patients undergoing noncardiac surgery, MINS is common (8%), one in ten patients suffering MINS will die within 30 days, and majority of these events can be only detected with hs-cTnT screening in the first 72 hours after surgery5. Therefore, failure to monitor troponin measurements after noncardiac surgery will result in missing more than 80% of MINS events4,5. In order to prevent missing of this prognostically relevant event and based on recommendations of some guidelines9,10, we will implement hs-cTnT screening programme within clinical routine. However, little is known about the feasibility and impact of hs-cTnT screening in real practice.\n\nOur study will implement routine hs-cTnT screening program, and evaluate its feasibility and impact at a tertiary hospital. Routine screening for perioperative hs-cTnT will lead to recognize most of MINS, improve clinical outcomes, and can potentially reduce short and long-term mortality after major non-cardiac surgery. Differently to previous studies, in case of elevated hs-cTnT a cardiologist will conduct a structured evaluation. Our hypothesis is that patients with a prognostically relevant hs-cTnT peak will likely improve their 1-month and 1-year prognosis if they receive structured management, treatment, and adequate follow-up. Clinicians will be better informed about how to interpret hs-cTnT values, and policy makers will be better informed to decide (or not) to implement routine troponin monitoring in high cardiovascular risk patients.\n\nPreoperative and postoperative hs-cTnT thresholds. In most of the previous studies hs-cTnT was obtained only after surgery4,5; however, in our study hs-cTnT will also be measured before surgery. This will help to determine the relevant pre and post-operative thresholds. There are only a few studies that have measured hs-cTnT levels before the time of surgery. The cohort study within the VINO trial (n = 608), concluded that hs-cTnT concentrations before surgery were significantly associated with postoperative MI, and long-term mortality after non-cardiac surgery8. We also identified a more recent cohort that measured hs-cTnT before surgery (within 30 days before surgery), where PMI was defined as an absolute increase in hs-cTnT of ≥14 ng/L above preoperative value, or between 2 postoperative values if the preoperative value was missing6. On the other hand, nearly half of adults undergoing non-cardiac surgery exceed the 99th percentile of ≥14ng/ post-surgery23, and mild elevations of hs-cTnT are common in men and elderly non-MI patients24, hence, optimal cut-off levels could differ across populations. We identified a single study showing cut-off values to differentiate acute MI from non-acute MI but in non-surgical elderly patients (>70 years old), being nearly four times the 99th percentile with hs-cTnT (54 ng/L). In contrast, the best cut-off value in younger patients was close to the 99th percentile for hs-cTnT (17 ng/L)25. Given the above, there is a need to determine optimal preoperative and post-operative prognostically relevant hs-cTnT thresholds in high cardiovascular risk patients undergoing major non-cardiac surgery.\n\nEconomic consequences. There are only few economic analyses studies evaluating the cost effectiveness of hs-cTnT monitoring, including a broad spectrum of non-cardiac surgical procedures. Despite the lack of conclusive economic evidence troponin monitoring is now recommended in some clinical guidelines9,10.\n\nWe identified two studies that analysed the cost-effectiveness of troponin monitoring11,12. The first study, conducted in USA, included patients aged ≥65 years undergoing open abdominal aortic aneurysm (AAA) repair11. The authors concluded that postoperative Troponin I screening after AAA repair was cost-effective, with an incremental cost-effectiveness ratio of 2003 US$12,641 per QALY. The second study was recently conducted by VISION study investigators. This is a model-based cost–consequence analysis which compares the impact of routine troponin T monitoring versus standard care on the incidence of MINS12. The model inputs were based on Canadian patients enrolled in VISION study. The costs associated with a troponin T monitoring program to detect MINS were moderate. The study concluded that implementation of troponin T monitoring seems appealing, particularly in patients at high risk for MINS, based on the estimated incremental cost per health gain. No cost-effectiveness analysis of hs-cTnT monitoring high-risk cardiovascular patients in major non-cardiac surgery patients (in comparison with usual care) is yet available. Our study will address this question in the Spanish setting using data from real clinical practice.\n\nMINS pathophysiology. Understanding the pathophysiology of MINS is crucial to develop potential prophylactic and therapeutic interventions to improve the prognostic of patients undergoing non-cardiac surgery. However, angiographic, histological or imaging studies that provide an overview of the incidence of etiological mechanisms of MINS are currently not available. We identified a single study where CTA was used for screening of coronary artery disease before non-cardiac surgery, improving perioperative risk stratification with clinical tools as the Lee index26. VISION study investigators have published a single prospective cohort study (Coronary CTA VISION) with 955 patients using CTA before non-cardiac surgery, concluding that myocardial infarction occurs across the spectrum of coronary artery disease, suggesting that there could be several pathophysiologic mechanisms involved27.\n\nGiven the scarce available data better understanding of the pathophysiology of MINS is much needed. With non-invasive diagnostic tools, such as CTA and MRI our study could improve the understanding of the underlying mechanisms involved in MINS. This new knowledge could inform prophylactic and therapeutic interventions.\n\nStudy limitations and strengths. Our study has several limitations. During hs-cTnT monitoring implementation, some monitored troponin measurements may be missed, due to fast hospital discharge, no staff collaboration, haemolysis of the samples, etc. Also there may have missed some structured cardiologist visits, due to fast hospital discharge or coordination problems. Therefore, we may miss MINS events in patients who do not experience ischemic symptoms. Regarding the cost-effectiveness study, there may be variability in sample-associated costs and results. Finally, the cardiac imaging sub-study has a nested case-control design, where inclusion of healthy controls without any clinical justification for carrying out diagnostic tests may be difficult.\n\nOur study has also several strengths. It is a rigorous proposal that will address several very relevant questions simultaneously. It will evaluate the feasibility and impact of implementing hs-cTnT monitoring program at a tertiary hospital, with large sample of adults who underwent noncardiac surgery. All patients will undergo troponin monitoring before and after surgery, using the same troponin assay. The study will also inform preoperative and post-operative prognostically relevant thresholds that likely will improve mortality and major cardiovascular events prediction. Our study will also determine the cost-effectiveness of troponin monitoring, and finally, the application of non-invasive advance imaging diagnostic tools (CTA and MRI), will contribute in the identification of the mechanisms involved in MINS.\n\nImplications for practice and research. The results of our cohort study will offer high-quality evidence to guide practice, and will likely have major implications in the management and prognosis of this public health problem. Success of implementation of the hs-cTnT monitoring program will be good example for its implementation in other sites, in Spain and elsewhere. New knowledge about preoperative and post-operative prognostically relevant thresholds will improve the prediction of mortality, and major cardiovascular events.\n\nGiven the low cost of hs-cTnT and poor prognosis of patients with MINS, potential management, treatment and hs-cTnT monitoring is likely to be cost-effective for the national health system. Our economic evaluation study will throw light on the cost effectiveness of troponin monitoring compared to usual care, which will likely improve prognosis and unnecessary costs. Policy makers will be better informed to decide (or not), to implement troponin screening in high cardiovascular risk patients.\n\nThe understanding of the pathophysiology of MINS will help to develop new prophylactic and therapeutic measures to improve the prognosis of patients, and reduce unnecessary costs. Moreover, the results of this study can help scientists to shape research initiatives in the future, applying these techniques to detection of myocardial injury. The success of this study will allow bringing research results to daily practice, evaluate their implementation, and facilitate further nested research that will address important questions in the field. This study is potentially an example of knowledge transfer, taking published results to real practice, with the aim to influence patient important outcomes.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nOpen Science Framework: Rationale and design of Perioperative Myocardial Ischemia (PMI): a protocol for troponin monitoring, prognostic thresholds, economic analysis and further insights into pathophysiology for non-cardiac surgery patients. https://doi.org/10.17605/OSF.IO/GBV4U19.\n\nOpen Science Framework: SPIRIT checklist for “Rationale and design of perioperative myocardial ischemia: a protocol for troponin monitoring, prognostic thresholds, economic analysis and further insights into pathophysiology for non-cardiac surgery patients”. https://doi.org/10.17605/OSF.IO/GBV4U19.", "appendix": "Grant information\n\nThis work was supported by the Fundació La Marató de TV3 (grant number: 20150110). PAC is supported by a Miguel Servet investigator contract from the Instituto de Salud Carlos III (CPII15/0034).\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\nEkaterine Popova is a doctoral candidate at the Paediatrics, Obstetrics and Gynaecology and Preventive Medicine Department, Universitat Autònoma de Barcelona, Barcelona, Spain. Catalonia, Spain: Iberoamerican Cochrane Centre. Biomedical Research Institute Sant Pau (IIB Sant Pau) working group: Francisco Rene Acosta, Raúl Aguilar Lopez and Mª Trinidad Muñoz Gallarin.\n\n\nReferences\n\nWeiser TG, Regenbogen SE, Thompson KD, et al.: An estimation of the global volume of surgery: a modelling strategy based on available data. Lancet. 2008; 372(9633): 139–44. PubMed Abstract \| Publisher Full Text\n\nLandesberg G, Beattie WS, Mosseri M, et al.: Perioperative myocardial infarction. Circulation. 2009; 119(22): 2936–2944. 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Health Expect. 2015; 18(6): 2318–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaniagua P, Popova E, Diaz R: European Journal of Anesthesiology. 2015; 32(Supplement 53): Abstract 8AP8–1.\n\nDescalzo M, Leta R, Rosselló X, et al.: Subclinical coronary atherosclerosis identified by coronary computed tomography angiography in asymptomatic population by coronary artery disease risk level. Rev Esp Cardiol (Engl Ed). 2013; 66(6): 504–505. PubMed Abstract \| Publisher Full Text\n\nKavsak PA, Walsh M, Srinathan S, et al.: High sensitivity troponin T concentrations in patients undergoing noncardiac surgery: a prospective cohort study. Clin Biochem. 2011; 44(12): 1021–1024. PubMed Abstract \| Publisher Full Text\n\nGore MO, Seliger SL, Defilippi CR, et al.: Age- and sex-dependent upper reference limits for the high-sensitivity cardiac troponin T assay. J Am Coll Cardiol. 2014; 63(14): 1441–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReiter M, Twerenbold R, Reichlin T, et al.: Early diagnosis of acute myocardial infarction in the elderly using more sensitive cardiac troponin assays. Eur Heart J. 2011; 32(11): 1379–89. PubMed Abstract \| Publisher Full Text\n\nHwang JW, Kim EK, Yang JH, et al.: Assessment of perioperative cardiac risk of patients undergoing noncardiac surgery using coronary computed tomographic angiography. Circ Cardiovasc Imaging. 2015; 8(3): pii:e002582. PubMed Abstract \| Publisher Full Text\n\nSheth T, Chan M, Butler C, et al.: Prognostic capabilities of coronary computed tomographic angiography before non-cardiac surgery: prospective cohort study. BMJ. 2015; 350: h1907. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "49799", "date": "20 Jun 2019", "name": "Christian Puelacher", "expertise": [ "Reviewer Expertise Clinical Research", "perioperative cardiology", "no Sound experience for economic analyses" ], "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 presented protocol seems very promising to help understand the clinical applicability of perioperative hs-cTnT Screening after non-cardiac surgery in high-risk patients in routine practise. The follow-up data on adherence of medication prescribed for MINS will be very interesting. The concept to do a cost-benefit analysis seems very promising as well. Also the CTA and MRI study is ambitious and could yield very interesting results.\nIt is important to note, that the study protocol was submitted in 05/2019 as the study nears completion (start according to the protocol was in 2016). Therefore, alterations to the original study protocol are possible, I would invite the authors to comment on any changes done especially concerning the endpoint or procedures.\nSome additional details could be considered:\nThe definition of MINS at the moment is to a part arbitrary as there is currently no agreed upon definition. There are however aspects to consider:\na) Previous studies were done, which used different cut-offs than that proposed by the study group. Importantly the VISION study identified possible cut-offs and the BASEL-PMI studies used a prospective cut-off. In both studies an absolute delta cut-off was used/found, which is in line with the agreed upon criteria for the definition of acute myocardial infarction. Using absolute delta cut-offs could enhance the protocol by creating a more readily usable flowchart (e.g. one cut-off for change to preoperative baseline) and allow for better comparison. Please consider doing a sensitivity analysis with aligned criteria.\nb) Using MINS as the primary outcome poses a challenge in the flow-Chart, as MINS excludes other conditions leading to postoperative troponin elevations, e.g. sepsis, pulmonary embolism or tachyarrhythmia. Do the authors wish to use the same distinction or include all elevations (which seems sensible, as this is what the screening will uncover)? Please elaborate.\n\nPrognostic Analysis: the combined MACE-endpoint consists of multiple endpoints, please extend the definition.\na) It is unclear to me why you ought to include unstable angina, as it is not expected that the coronary artery disease of the affected patients should worsen via surgery? This could introduce noise into this ambitious study\nb) New atrial fibrillation will be difficult to assess using only one ECG at detection. Would you reconsider \"onset of symptomatic AF or AF needing treatment\"?\nc) Why is \"cardiovascular death\" not part of the combined endpoint?\nd) The use of a binary logistic model instead of a Cox proportional hazards model or a time-dependent analysis is unclear to me. As a certain amount of loss-to-followup is nearly unavoidable, and there is a competing endpoint all-cause death or (in case of cv-death being part of the endpoint) non-cardiovascular death, the Cox-model would offer benefits.\n\nWhen evaluating potential threshold for the prognostic ability of hs-cTnT/MINS, I would recommend a methodology similar to that of the VISION study, which was done in a very thorough manner.\n\nSubstudy: Could you provide details on the matching procedure done for the substudy? Is this propensity score matching?\n\nCost-benefit analysis: in light of no clear MINS-Definition to date, I invite the authors to do sensitivity analyses using different assumptions for the definition of MINS.\n\nThe provided ideas are simply comments, the authors shall feel free to use any recommendations, especially as the study was already conducted according to the methods. I congratulate the research group for such an undertaking.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4789", "date": "02 Aug 2019", "name": "Ekaterine Popova", "role": "Author Response", "response": "Dear Dr. Puelacher,Thank you very much for substantial revision and useful comments and suggestions. However, at this moment only one peer reviewer report has been received, so we can not submit a final revised version until additional comments have been received from another reviewer.Please see our responses below.Christian PuelacherDepartment of Cardiology, University Hospital of Basel, University of Basel, Basel, SwitzerlandThe presented protocol seems very promising to help understand the clinical applicability of perioperative hs-cTnT screening after non-cardiac surgery in high-risk patients in routine practice. The follow-up data on adherence of medication prescribed for MINS will be very interesting. The concept to do a cost-benefit analysis seems very promising as well. Also the CTA and MRI study is ambitious and could yield very interesting results.It is important to note, that the study protocol was submitted in 05/2019 as the study nears completion (start according to the protocol was in 2016). Therefore, alterations to the original study protocol are possible; I would invite the authors to comment on any changes done especially concerning the endpoint or procedures.Reply: there have been no major changes in the protocol except for the inclusion of a new center to increase the recruitment for the sub-study 3 (ACE-CARD), and a modification in the MACCE definition. We have included a sentence in the protocol to reflect this in the manuscript (section “Current status of the trial”, line 3). It reads as follows:“Since the start of the study in 2016 there have been two modifications in the original protocol; one new center (Hospital Vall d’Hebron) joined the ACE-CARD substudy and the MACCE definition was modified, excluding unstable angina. The reason for including a new center was the low recruitment rate. The MACCE definition was modified to according to the European perioperative clinical outcome (EPCO) definition statement (Jamer Ib, 2015).”Ref.: Jamer Ib, Wickboldt N, Sander M, Smith A, Schultz MJ, Pelosi P, et al. Standards for definitions and use of outcome measures for clinical effectiveness research in perioperative medicine: European perioperative clinical outcome (EPCO) definitions. Eur J Anesthesiol 2015;32: 88-105.Some additional details could be considered: The definition of MINS at the moment is to a part arbitrary as there is currently no agreed upon definition. There are however aspects to consider:a) Previous studies were done, which used different cut-offs than that proposed by the study group. Importantly the VISION study identified possible cut-offs and the BASEL-PMI studies used a prospective cut-off. In both studies an absolute delta cut-off was used/found, which is in line with the agreed upon criteria for the definition of acute myocardial infarction. Using absolute delta cut-offs could enhance the protocol by creating a more readily usable flowchart (e.g. one cut-off for change to preoperative baseline) and allow for better comparison. Please consider doing a sensitivity analysis with aligned criteria. Reply: thank you for the suggestion. We will perform sensitivity analysis considering absolute delta cut-offs instead of relative changes.The section now reads:\"We will assess the agreement between diagnostic findings in the MINS population and the control group using Cohen’s kappa coefficient. We will conduct sensitivity analysis using absolute delta cut-offs to define MINS in accordance to previous studies (Devereaux PJ, 2012; Puelacher C, 2018). For the significance level we will use a 5% (alpha = 0.05) bilateral approach.\"Ref.: Devereaux PJ, Chan MT, et al. Vascular Events In Noncardiac Surgery Patients Cohort Evaluation (VISION) Study Investigators, Association between postoperative troponin levels and 30-day mortality among patients undergoing noncardiac surgery. JAMA. 2012; 307(21):2295–304.Ref.: Puelacher C, Lurati Buse G, Seeberger D, BASEL-PMI Investigators. Perioperative Myocardial Injury after Noncardiac Surgery: Incidence, Mortality, and Characterization. Circulation. 2018 Mar 20; 137(12):1221-1232. doi: 10.1161/CIRCULATIONAHA. b) Using MINS as the primary outcome poses a challenge in the flowChart, as MINS excludes other conditions leading to postoperative troponin elevations, e.g. sepsis, pulmonary embolism or tachyarrhythmia. Do the authors wish to use the same distinction or include all elevations (which seems sensible, as this is what the screening will uncover)? Please elaborate. Reply: we considered the definition of MINS (VISION study), as an elevated troponin measurement that occurs between surgery and the first 30 days after surgery, judged as resulting from myocardial ischemia (i.e., no evidence of a non-ischemic etiology such as pulmonary embolism, atrial fibrillation or sepsis). This definition includes patients with MI and patients with troponin elevation due to ischemic causes that don’t fulfill criteria of conventional myocardial infarction (Thygesen K 2018).Ref.: Thygesen K, Alpert JS, Jaffe AS, et.al. Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction. Glob Heart. 2018 Aug 23. pii: S2211-8160(18)30138-8. doi: 10.1016/j.gheart.2018.08.004. Prognostic Analysis: the combined MACE-endpoint consists of multiple endpoints, please extend the definition. Reply: we have modified the MACCE definition according the European perioperative clinical outcome (EPCO) definitions statement (Jamer Ib, 2015). Please, see Appendix 1. a) It is unclear to me why you ought to include unstable angina, as it is not expected that the coronary artery disease of the affected patients should worsen via surgery? This could introduce noise into this ambitious study Reply: unstable angina should not have been included in the composite. We have modified accordingly. b) New atrial fibrillation will be difficult to assess using only one ECG at detection. Would you reconsider \"onset of symptomatic AF or AF needing treatment\"? Reply: we have modified the terminology to address this concern. We are now using “new clinically important atrial fibrillation” instead of “new atrial fibrillation.” c) Why is \"cardiovascular death\" not part of the combined endpoint? Reply: we consider all-cause death as the primary outcome. It is a more patient important outcome than the outcomes included in the composite, and we prefer to keep it separate. Nevertheless, we will report both vascular and non-vascular death. d) The use of a binary logistic model instead of a Cox proportional hazards model or a time-dependent analysis is unclear to me. As a certain amount of loss-to-follow up is nearly unavoidable, and there is a competing endpoint all-cause death or (in case of cv-death being part of the endpoint) non-cardiovascular death, the Cox-model would offer benefits. Reply: we agree about using a Cox model instead of logistic regression. We will analyze time-to-death and time-to-MACCE using proportional hazard models. We will explore factors associated with death and MACCE by including clinically relevant variables into the multivariate models. We will obtain a final MACCE risk model following a backward elimination strategy. We will assess the proportional hazard assumption of all factors included in the analysis using Schoenfeld residuals plots. We will adjust for overoptimism in model performance by bootstrap validation. We will assess the overall discriminatory ability of the model using the C-statistic. We will evaluate model calibration using calibration plots, and we will compute the calibration slopes. When evaluating potential threshold for the prognostic ability of hs-cTnT/MINS, I would recommend a methodology similar to that of the VISION study, which was done in a very thorough manner. Reply: we will use two methodologies, including the one you suggest despite it is more related to the determination of the hs-cTnT cut-off point in the context of a multivariate predictive model. Our goal is to identify \"univariate\" cut-off points. We have also included a new reference (Mazumdar 2003), section “Statistical analysis”, line 20.The section now reads:“Finally, from receiver-operator characteristic (ROC) curve analysis, we will univariately estimate the best pre and post hs-cTnT cut-off values to predict 30 days and 1 year after surgery mortality and MACCE. We will select the cut-off that maximizes Youden’s index, and secondary cut off values to achieve sensitivities of 80, 85, 90, and 95%. For the predictive modeling, we will also explore what pre and post-surgery hs-cTnT threshold values will be better predictors to be used in the predictive multivariate models, for 30 days and 1 year after surgery mortality and MACCE. For this, we will use an iterative process based on Mazumdar et al methodology (Mazumdar 2003). In short, we will cross-validate different hs-cTnT thresholds and select the optimal cut-off based on log-likelihood tests of the predictor within the multivariate model.”Ref.: Mazumdar M, Smith A, Bacik J. Methods for categorizing a prognostic variable in a multivariable setting. Stat Med. 2003; 22(4):559–71. Substudy: Could you provide details on the matching procedure done for the substudy? Is this propensity score matching? Reply: patients in the ACE-CARD substudy will be simple matched by sex, age (within five years of an interval), type of surgery, and Revised Cardiac Risk Index (Lee Criteria). Cost-benefit analysis: in light of no clear MINS-Definition to date, I invite the authors to do sensitivity analyses using different assumptions for the definition of MINS. Reply: thank you for the suggestion. We will conduct a sensitivity analysis using several definitions of MINS. We would like to clarify that we plan to conduct a cost-effectiveness analysis and not a cost-benefit analysis.The provided ideas are simply comments; the authors shall feel free to use any recommendations, especially as the study was already conducted according to the methods.I congratulate the research group for such an undertaking.Appendix 1.MACCE (Major Adverse Cardiac and Cerebrovascular Events) is defined as one or more of the following: Myocardial injury after noncdiac surgery (MINS):1.1. Myocardial infarction MI (3rd universal definition):The diagnosis of MI requires at least one of the followings: Detection of a rise and/or fall of a cardiac biomarker (preferably troponin) with at least one value above the 99th percentile of the upper reference limit (URL) together with evidence of myocardial ischemia with at least one of the followings: a) signs or symptoms of ischemia (i.e., chest, arm, neck, or jaw discomfort; shortness of breath, pulmonary edema); b) new or presumed new ECG changes indicative of ischemia (i.e., ST segment elevation [≥ 2 mm in leads V1, V2, or V3 OR ≥ 1 mm in the other leads], ST segment depression [≥ 1mm], or symmetric inversion of T waves ≥ 1 mm) in at least two contiguous leads; c) new left bundle branch block (LBBB); d) development of pathological Q waves present in any two contiguous leads that are ≥ 30 milliseconds; d) new cardiac wall motion abnormality on echocardiography or new fixed defect on radionuclide imaging; or e) identification of an intracoronary thrombus by angiography or autopsy.1.2. Myocardial injury after noncardiac surgery (MINS) not meeting 3rd universal definition of MI:For MINS diagnosis following criterion is required: detection of a rise or fall of Troponin with at least one value above the 99th percentile of the upper reference limit (URL) AND 50% higher than the previous troponin measurement (before surgery), related to the preceding event that is judged as resulting from myocardial ischemia (i.e., no evidence of a non-ischemic etiology causing the troponin elevation such as pulmonary embolism or sepsis) AND does not fulfil the myocardial infarction definition above. Non-fatal cardiac arrest, defined as an absence of cardiac rhythm or presence of chaotic rhythm requiring any component of basic or advanced cardiac life support. Congestive heart failure, The definition of new congestive heart failure requires a clinical sign (i.e. at least one of the followings: elevated jugular venous pressure, respiratory rales/crackles, crepitations, or presence of S3) and a radiographic finding (i.e., at least one of the followings: vascular redistribution, interstitial pulmonary edema, or frank alveolar pulmonary edema). New clinically important atrial fibrillation, is defined as important atrial fibrillation, documented with ECG evidence of atrial flutter, atrial fibrillation, or second- or third-degree atrioventricular conduction block, and results in angina, congestive heart failure, symptomatic hypotension, or requires treatment with a rate controlling drug, antiarrhythmic drug, or electrical cardioversion. Stroke, is defined as a new focal neurological deficit thought to be vascular in origin with signs or symptoms lasting more than 24 hours or leading to death" } ] }, { "id": "126837", "date": "28 Mar 2022", "name": "Sebastiano Gili", "expertise": [ "Reviewer Expertise acute coronary syndromes", "coronary artery disease", "interventional cardiology", "post-pci myocardial injury" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPopova and colleagues present the rationale and design for a prospective study aiming to assess the clinical implications and cost-effectiveness of a structured program for the monitoring of pre- and post-operative troponin values in patients undergoing high-risk surgery. The main aim of the study is to evaluate the incidence of myocardial injury after non-cardiac surgery (MINS) and to explore its pathophysiology.\nThe presented protocol is of interest and the implementation of the study will be able to provide useful clinical information. There are some issues however that need to be discussed:\nPlease provide definitions for documented cardiovascular disease in patients < 65 years old, in particular for coronary artery disease, peripheral artery disease and chronic heart failure.\n\nPost-surgical troponin elevation management is susbtantially left to the clinical decision and judgement of the cardiologist in charge of evaluating the patient. This could introduce some inconsistencies in the treatment of patients, the authors should consider to provide broad guidelines in this sense.\n\nC-statistic and ROC curve analysis will be performed on a relatively low number of expected events (about 150 expected MINS) and no internal or external validations is contemplated in the study protocol.\n\nCost-effectiveness analysis: it is not clear how the comparator for this analysis, and in particular for the assessment of QALY, will be adopted for this anlaysis.\n\nThe authors should clarify how they consider a simple score such as the Lee score, with well-known limitations, will be able to provide adequate matching in terms of cardiovascular risk and history in patients undergoing imaging studies.\n\nGiven that the study is centered in the assessment of clinical implications of MINS, please discuss why the authors opted to include also overt post-operative MI in the imaging analysis.\n\nPlease discuss how incidental findings of subclinical coronary artery disease will be managed in the patients included in the imaging substudy, in particular in those without MINS/MI.\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/8-850
https://f1000research.com/articles/8-288/v1	14 Mar 19	{ "type": "Research Note", "title": "Examining the role of funders in ensuring value and reducing waste in research: An organizational case-study of the Patient-Centered Outcomes Research Institute", "authors": [ "Evelyn P. Whitlock", "Joe V. Selby", "Kelly M. Dunham", "Alicia Fernandez", "Laura P. Forsythe", "Grayson Norquist", "Joe V. Selby", "Kelly M. Dunham", "Alicia Fernandez", "Laura P. Forsythe", "Grayson Norquist" ], "abstract": "International experts have recommended actions that funders can take to improve the value of research investments. They state that self-assessment and public sharing are the basis for accountability and improvement. We examined our policies and practice to determine the extent to which the Patient-Centered Outcomes Research Institute’s (PCORI) policies and practices as a research funder align with international best practice recommendations. A self-audit of current policies and practice against 17 recommendations and 35 sub-recommendations representing five major stages of research production, based on adapted methods used for self-assessment by another funder, was performed. Fit of existing PCORI policies and practices with 35 sub-recommendations, qualitative assessment of adequacy (area of strength; area of partial strength; area of growth; not applicable) for 17 recommendations for five stages of research production was assessed. Of the 17 recommendations, 15 were applicable to PCORI’s research mission and focus. PCORI has policies and practices in place for all elements of six recommendations (“area of strength”) and policies that address each element but with some still in active development for three (“area of partial strength”). PCORI is partially addressing six of the 15 relevant recommendations (“area of growth”). Areas for growth include making study protocols publicly available, improving policies on data sharing, and enhancing collaboration with other funders to reduce redundant funding. A voluntary consortium of international funders is underway to encourage further progress, including additional self-assessment and public sharing for accountability. These findings indicate PCORI has undertaken efforts to align its funding practices with international recommendations to ensure the value of public dollars invested in research. Further efforts will likely require additional coordination and collaboration between funders and stakeholders.", "keywords": [ "Biomedical Research/standards", "Research Design/standards", "Biomedical Research/economics", "Biomedical Research/methods" ], "content": "Introduction\n\nIn 2014, in response to concerns about avoidable waste in research prioritization, conduct, and reporting1, The Lancet published a series of articles which identified specific recommendations for the biomedical research community to ensure value and minimize inefficiency in research2–6. Research funders were a major target for these recommendations, along with regulators, journals, academic institutions and researchers themselves. Prompted by these and related activities, the biomedical research community around the world has begun considering best practices to ensure value in publicly-funded research. As key contributors7, research funders are encouraged to audit and update their own policies and practice, even as external assessments of funders are also undertaken8,9.\n\nIn light of these trends, the Patient-Centered Outcomes Research Institute (PCORI), undertook an organizational case study of its policies and practices. PCORI was created in 2010 to address research needs of a range of healthcare stakeholders through clinical comparative effectiveness research, and ranks among the top 10 US non-commercial funders of health research (see Healthresearchfunders.org). Our goals were to examine and report how closely PCORI adheres to best practice recommendations for research funders (i.e., to foster transparency), to highlight areas of needed development for PCORI (to foster public accountability), and to consider how other research funders in the US and elsewhere can examine, report, and adopt best practices for supporting value in research (to foster enterprise-wide efficiency).\n\n\nMethods\n\nTo maximize comparability, we adapted another funder’s self-assessment methods (See Adding Value to Research from the National Institute for Health Research). PCORI staff (KD, LF, EW) examined PCORI’s existing policies and initiatives against 17 recommendations for funders, using a total of 35 sub-recommendations to capture multiple dimensions within some recommendations (see PCORI site). Four authors (KD, LF, EW, GN) independently categorized fidelity to the 17 recommendations as: 1) “area of strength” –PCORI’s practices reasonably address all sub-recommendations; 2) “area of partial strength” –PCORI’s practices reasonably or partially address all sub-recommendations; 3) “area of growth” –PCORI’s practices do not address all sub-recommendations, either reasonably or partially; or 4) not applicable. We resolved discrepancies through discussion and final ratings reflect consensus.\n\n\nResults\n\nTable 1 represents a detailed summary (through November 2018) of PCORI’s policies and practices related to ensuring value in research. Across the 17 recommendations (35 sub-recommendations), two recommendations were not applicable (1, 8), and one recommendation primarily applies to non-funders (both 9a, 9b). For the 15 relevant recommendations, PCORI at least partially addresses most of the relevant sub-recommendations (28/33). Our consensus process categorized PCORI’s existing policies and practices as “areas of strength” for 6/15 applicable recommendations, “partial strength” for 3/15. PCORI’s authorizing legislation, although preceding the Lancet recommendations by several years, mandated a number of these (indicated in bold in the table).\n\nRecommendations sourced from Lancet series on ensuring value and minimizing waste in research2–6\n\nBolded text indicates process or policy mandated by PCORI’s authorizing legislation\n\n\nDiscussion\n\nOur consensus process categorized PCORI’s existing policies and practices as meeting criteria for “areas of strength” or “partial strength” for many of the recommendations, and we also identified clear areas for growth. Examples of strengths include PCORI’s requirements that funded research adhere to methodology standards to minimize bias and that all study results are posted on the PCORI website to enhance public access to findings. On the other hand, PCORI has not yet fully developed its policies and practices related to rewarding research replication and reproducibility (Recommendation 7). Further development of performance metrics, standardized approaches to all study-related reporting, and enforcement of key policies (Recommendations 12, 13, 14) offer other areas ripe for growth, particularly if undertaken in coordination with others across the research enterprise. PCORI like many funders, is still actively developing its practices related to publicly sharing information, including raw data, as early as possible from funded research (Recommendations 4, 5). For example, making research protocols publicly available (Recommendation 5a) is required by PCORI’s authorizing legislation, but timing and format were not specified, and our current practices may not be ideal. PCORI now requires funded investigators to submit a study protocol and record its details in an appropriate registry but does not yet specify a standard protocol format nor require protocol publication before study completion. To our knowledge, just one funder (NIHR) clearly publishes study protocols at the time of award10. Nonetheless, making study protocols available at study inception can benefit the public by providing a detailed record of the planned study, which may help avoid unwitting duplication of research underway and support detection of important study deviations and post-hoc changes.\n\nThere is also opportunity for improvement through further development of policies and practices related to research data sharing and re-use. While funders can require awardees to share data from funded research and trial participants are supportive of such sharing11, many researchers remain concerned about the impact on their work12. PCORI’s policy on data sharing13 was informed by a public comment process as well as pilot work assessing time and effort required for investigators to prepare their data for sharing and on identifying appropriate repository models. Accelerating the practice of responsible data sharing necessitates broad coordination between journals, academic institutions, and data-repository organizations, alongside consistent requirements and support from funders.\n\nEfforts to reduce waste and increase value in research are in alignment with trials transparency14, research integrity15, administrative efficiency16, and other similar initiatives. PCORI and other health research funders are in consortium to encourage further development and voluntary adherence to international best practice recommendations for research funders, (17; see Ensuring Value in Research (EVIR) website). The Ensuring Value in Research Funders’ Forum is exploring other initiatives, such as evaluating and sharing best practices for similar challenges that funders face, and considering what avenues exist to enhance efficiency and value in the full research agenda across funders. Beyond the consortium, greater transparency and coherence between funders and key players producing health research---including journals, research institutions, sponsors, and regulators---remains vital for tangible progress in our shared efforts7,18.\n\nLimitations: Our methods are limited by self-assessment, but findings are consistent with audit results for PCORI from external assessors10. In addition, the availability of policies or current practices represent only the first step, with actual performance measurement needed. Finally, while the Lancet series highlights areas for improvement for funders and others across the research enterprise, the impact of implementing and adhering to these recommendations on research value has yet to be demonstrated.\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\nThe authors thank Hal Sox, MD, for comments that greatly improved the manuscript.\n\n\nReferences\n\nChalmers I, Glasziou P: Avoidable waste in the production and reporting of research evidence. Lancet. 2009; 374(9683): 86–9. PubMed Abstract \| Publisher Full Text\n\nAl-Shahi Salman R, Beller E, Kagan J, et al.: Increasing value and reducing waste in biomedical research regulation and management. Lancet. 2014; 383(9912): 176–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChalmers I, Bracken MB, Djulbegovic B, et al.: How to increase value and reduce waste when research priorities are set. Lancet. 2014; 383(9912): 156–65. PubMed Abstract \| Publisher Full Text\n\nChan AW, Song F, Vickers A, et al.: Increasing value and reducing waste: addressing inaccessible research. Lancet. 2014; 383(9913): 257–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlasziou P, Altman DG, Bossuyt P, et al.: Reducing waste from incomplete or unusable reports of biomedical research. Lancet. 2014; 383(9913): 267–76. PubMed Abstract \| Publisher Full Text\n\nIoannidis JP, Greenland S, Hlatky MA, et al.: Increasing value and reducing waste in research design, conduct, and analysis. Lancet. 2014; 383(9912): 166–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKleinert S, Horton R: How should medical science change? Lancet. 2014; 383(9913): 197–8. PubMed Abstract \| Publisher Full Text\n\nWhitlock EP, Dunham KM, DiGioia K, et al.: Noncommercial US Funders' Policies on Trial Registration, Access to Summary Results, and Individual Patient Data Availability. JAMA Netw Open. 2019; 2(1): e187498. PubMed Abstract \| Publisher Full Text\n\nDeVito NJ, French L, Goldacre B: Noncommercial Funders' Policies on Trial Registration, Access to Summary Results, and Individual Patient Data Availability. JAMA. 2018; 319(16): 1721–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNasser M, Clarke M, Chalmers I, et al.: What are funders doing to minimise waste in research? Lancet. 2017; 389(10073): 1006–7. PubMed Abstract \| Publisher Full Text\n\nMello MM, Lieou V, Goodman SN: Clinical Trial Participants' Views of the Risks and Benefits of Data Sharing. N Engl J Med. 2018; 378(23): 2202–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiley R, Peatfield T, Hansen J, et al.: Data Sharing from Clinical Trials - A Research Funder's Perspective. N Engl J Med. 2017; 377(20): 1990–2. PubMed Abstract \| Publisher Full Text\n\nPatient-Centered Outcomes Research Institute (PCORI): Policy for Data Management and Data Sharing. Accessed February 21, 2019. Reference Source\n\nHudson KL, Lauer MS, Collins FS: Toward a New Era of Trust and Transparency in Clinical Trials. JAMA. 2016; 316(13): 1353–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Academies of Sciences, Engineering, and Medicine: Fostering Integrity in Research. Washington, DC: The National Academies Press; 2017. Publisher Full Text\n\nOffice USGA: Federal Research Grants: Opportunities Remain for Agencies to Streamline Administrative Requirements. 2016. Reference Source\n\nChinnery F, Dunham KM, van der Linden B, et al.: Ensuring value in health-related research. Lancet. 2018; 391(10123): 836–7. PubMed Abstract \| Publisher Full Text\n\nGlasziou P, Chalmers I: Research waste is still a scandal-an essay by Paul Glasziou and Iain Chalmers. BMJ. 2018; 363: k4645. PubMed Abstract \| Publisher Full Text" }	[ { "id": "47387", "date": "25 Apr 2019", "name": "Hans Lund", "expertise": [ "Reviewer Expertise My professional content area is research within rehabilitation. Methodologically", "I am using systematic reviews", "meta-analyses", "and meta-research. As the chair of the \"Evidence-Based Research Network\" I am fully occupied with issues related to promote ways of thinking and acting to improve the quality of research and to avoid waste in 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 study is a report of a self-audit done by one of the top 10 non-commercial funders in USA. The aim was to evaluate to what degree the funding agency follow international recommendations to improve the value of research investments. This self-audit is very important for a general audience as:\nResearchers can understand the context and environment of funding and the reason for the requirements related to application for funding Other funding agencies can see how to change their policies in order to improve the value of research investments Readers will understand the challenges related to improve the value of research investments\nThe report should include a date for when to expect an update of the self-audit. An update - for example 2 to 3 years from now - would show the improvements and identify the biggest challenges related to improve the value of research investments.\nThe method is only partly described as the reader is unable to see from where the 17 recommendations and 35 subrecommendations originates. None of the link leads the reader directly to the source. In addition, as the authors use another funder´s self-assessment method, the possible alterations or adjustments made in the present self-audit should be mentioned. If no alterations were done, this should also be mentioned. Using the same assessment method makes is possible to compare, and this could have been mentioned in the Discussion.\nThere is an * and a † in Table 1, I can´t find what these refers to.\nIn conclusion: this is a very important and useful report of a self-audit (see above), and with the minor adjustments mentioned is should be published the sooner the better.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4668", "date": "11 Jun 2019", "name": "Kelly Dunham", "role": "Author Response", "response": "We greatly appreciate the reviewer's comments and suggestions. While we routinely monitor PCORI practice, we plan to conduct a second self-assessment in two years. We added this information to the discussion section.We appreciate the reviewer's suggestion regarding the methods section. In the revised article, we explicitly linked to the 17 recommendations from the Lancet series. We made clearer in the text that we broke these into a total of 35 sub-recommendations, since the original 17 sometimes included multiple items against which it would have been difficult to assess and transparently communicate our current activities. In other words, it would have been easy to claim credit for doing one part of a recommendation when we were not addressing another aspect of the recommendation at all. The other funder's self-assessment was not published, so it cannot be linked to directly. We added a reference to personal communication in the revised article. We did not have a copy of their written methods so cannot make the clear comparison that is requested, but have made our approach more transparent to avoid confusion.Thank you for pointing out the miscellaneous symbols in the table. These were holdovers from a previous version of the table and have been removed. Thank you for the thoughtful critique. Your comments and questions have helped us to clarify the methods used and greatly improved the article." } ] }, { "id": "45788", "date": "14 May 2019", "name": "Mona Nasser", "expertise": [ "Reviewer Expertise Clinical Epidemiology (focusing on priority setting and how funders allocate funding for research and systematic reviews)" ], "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 project and the authors have put a lot of effort to ensure a structured approach to self-evaluation of the organisation.\n\nIt would be helpful to have more details on the methods:\nDid all four authors go through all questions and sections, or was it divided between them with at least two people independently having seen each section? Did individuals respond to the question based on their experience or knowledge, or did it additionally involve any review of policies and documents internally by them? I was not sure what you meant by “reasonably” – did you mean that PCORI has generally met the recommendation, even if there is room for improvement available (unlike “area of growth” which is a gap and needs improvement). The table provides a very good summary but in some cases like area 4 and 12, it isn’t clear where the areas of growth are, so would be helpful to explain it (which is the part I mentioned is missing from the data to be able to reproduce).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4669", "date": "28 May 2019", "name": "Kelly Dunham", "role": "Author Response", "response": "We greatly appreciate the reviewer's questions and suggestions. We made several revisions to the article in response to the comments. At least two PCORI staff members reviewed each recommendation. Policies and documents were reviewed, where applicable, in addition to knowledge-based documentation. Our assessments were confirmed by other staff who were not involved as co-authors, particularly for policies and procedures not centrally documented. We appreciate the reviewer's question about the meaning of \"reasonably.\" We employed the term \"reasonably\" to indicate two things. First, that PCORI has generally met the recommendation. Second, that any assessment made relies on a qualitative judgement, but one that we sought to measure against a standard of reasonableness, i.e., \"reasonable people would come to the same conclusion looking at the same information.\" Thank you for this question, which allows us to provide this further clarification. We appreciate the request to be explicit, and made an effort to do so with examples in the text for areas like making the protocol publicly available (Recommendation 5a) and data sharing (Recommendations 12-14). We are limited in our ability to be completely explicit across all areas of growth for several reasons: 1) in some areas, such as ensuring the complete availability and sharing of primary study data, practices for funders and other actors are less established and more developmental than in other domains. We don't have clear standards against which to assign specific areas of growth; and 2) some areas of growth require strategic decisions and direction by governing bodies, once identified. For these areas, we cannot articulate next steps without a more deliberative process which this audit and resultant activities should help stimulate. Thanks again for the thoughtful critique; your comments have greatly improved the article." } ] } ]	1	https://f1000research.com/articles/8-288
https://f1000research.com/articles/8-845/v1	11 Jun 19	{ "type": "Research Article", "title": "Assessment of cardiovascular risk in post-menopausal women in Ghana", "authors": [ "Justice Afrifa", "Felix A. Botchway", "Yeboah Kwaku Opoku", "Joyce Badohu", "Henrietta Ekua Ocran", "Kwame Kumi Asare", "Samuel Essien-Baidoo", "Justice Afrifa", "Felix A. Botchway", "Joyce Badohu", "Henrietta Ekua Ocran", "Kwame Kumi Asare", "Samuel Essien-Baidoo" ], "abstract": "Background: Cardiovascular diseases (CVD) continue to be a major cause of death among post-menopausal women. We sought to assess cardiovascular risk among pre- and post-menopausal women living within the Cape Coast Municipality by comparing the lipid profiles and other emerging biomarkers of CVD, i.e. the atherogenic index of plasma (AIP), visceral adiposity index (VAI), body adiposity index (BAI) and Castelli index I (CRI-I). Methods: A comparative cross-section of 150 women (75 pre-menopausal women and 75 post-menopausal women) visiting the University of Cape Coast hospital for regular checkups were randomly recruited into the study. Socio-demographic and clinical characteristics of participants were obtained with the aid of a structured questionnaire. Blood pressure (BP) was measured and lipid profile was estimated using fasting blood samples. Other markers of cardiovascular risk such as BMI, AIP, VAI, BAI and CRI-I were estimated. Results: We report elevated levels of total cholesterol (TC) (p<0.0001), low density lipoprotein (LDL) (p<0.0001), very low-density lipoprotein (VLDL) (p=0.0021), triglycerides (TG) (p<0.0001) and non-high-density lipoprotein (non-HDL-C) cholesterol (p<0.0001) in post-menopausal women compared with pre-menopausal women. High-density lipoprotein (HDL) (p<0.0001) was, however, decreased in post-menopausal women. Mean AIP (p< 0.0001), VAI (p< 0.0001), BAI (p< 0.0038) and CRI-I (p<0.0001) were significantly increased in post-menopausal women compared to pre-menopausal women. We also report a positive correlation of TC, TG, VLDL and non-HDL with atherogenic markers AIP, VAI and CRI-I in post-menopausal women. A negative correlation of HDL with AIP, VAI, and CR in post-menopausal women was also observed. Conclusions: Menopause could lead to changes in lipid profile to atherogenicity with associated increase in the risk of CVD. Atherogenic markers such as AIP, VAI, BAI, and CR can serve as potential biomarkers for predicting CVD.", "keywords": [ "Atherogenicity", "menopause", "Lipid profile", "cardiovascular diseases" ], "content": "Introduction\n\nMenopause is a permanent physiological state with cessation of menstruation attributable to the loss of ovarian function and reduction in the production of estrogen1. The average age of menopause is reported to be 51 years2 but the age of natural menopause may vary from 40 to 58 years. This phase is characterized by variety of changes in socio-cultural, physiological and psychological states. These changes culminate into myriad of symptoms including insomnia, sweating, hot flashes, depressive mood, vaginal dryness and general discomfort3.\n\nCardiovascular diseases (CVD) are the major cause of death among post-menopausal women4. Studies have shown that pre-menopausal women have a low risk of CVD as compared to men but after menopause the level of risk increases5. Epidemiological data have revealed elevated risk of CVD in post-menopausal females compared to men of the same age6. Estrogen is the major female hormone that regulates many aspects of a female’s development. Reduced circulating estrogen levels impeded ovulation as results of under stimulation of the hypothalamus to release follicle stimulating hormones7,8. Estrogen is known to possess both anti-atherogenic and cardioprotective effect by maintaining high levels of high-density lipoprotein (HDL-C) coupled with decreasing low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG)9. Several factors, including diabetes, hypertension, and atherosclerosis among others can lead to CVD in women. The World Health Organization (WHO) has ranked CVDs as the number one cause of death, with global estimation of about 17.7 million deaths in 201510.\n\nCurrent data have shown that Castelli risk index I and II which are estimated as TC/HDL-C and LDL/HDL-C ratios respectively predict cardiovascular risk accurately than conventional lipid profile indices such as serum TC and serum triglycerides11,12. Similarly, comparison of individual lipid ratios in subjects of the Framingham Heart Study unarguably indicates that lipid ratios are significantly more useful predictors of CVD than the individual levels of LDL or HDL12. Many clinical studies have also made efforts to introduce better markers of atherogenic dyslipidemia that can predict the risk of CVD to be useful for evaluating response to treatment instead of the classical ratios13. Notable among these markers is the atherogenic index of plasma, which has proven to be a strong marker for the prediction of atherosclerosis and coronary heart disease risk14.\n\nMenopausal health has received little attention due to high illiteracy rate, poverty and a possible lack of understanding of the clinical dynamics associated with it, irrespective of the fact that menopause is a key risk factors for dyslipidemia which affects cardiovascular health of women. Compared with men, the diagnosis of CVD in women is difficult due to hormone-related changes and difference in sex in the clinical manifestation of CVD. Such limitations and challenges present the need for an increased consciousness of the significance of CVD as a major public health issue for older women. This study therefore sought to assess the cardiovascular risk between pre and post-menopausal woman in the Cape Coast municipality by comparing the lipid profiles and other emerging biomarkers of CVD such as atherogenic index of plasma visceral adiposity index and basal adiposity index.\n\n\nMethods\n\nWe conducted this comparative cross-sectional study among a convenience sample of 150 women between June 2017 and August 2018 at Cape Coast, the capital of the Central Region of Ghana. No attempts were made to control for bias in recruitment. This Region covers an area of approximately 9826 square kilometres or 4.1% of Ghana’s land area. The study participants were apparently healthy pre- and post-menopausal women.\n\nThe inclusion criterion used for the study was healthy postmenopausal women aged 40–55 years serving as cases while premenopausal women within the age of 30–40 years were used as comparative controls.\n\nThose with known cardiovascular and metabolic diseases such as hypertension, diabetes, renal or hepatic disorders, menstrual disorders and those on hormonal replacement therapies were excluded from the study. Also, pregnant and lactating women, women with known thyroid diseases, heavy smokers and alcoholics were not included in the research.\n\nEthical approval for the research was sought from the Institutional Review Board of University of Cape Coast (UCCIRB/CHAS/2017/83). All information regarding the study including the purpose, risks, procedures, and benefits were made known to the participants before seeking for written informed consent.\n\nFrom each participant, 4 mL of venous blood samples were collected from each participant with a sterilized syringe and needle after an overnight fast (12–14 hours) and dispensed into a gel separator tube. Blood samples were analyzed using a fully automated chemistry analyzer (Mindray BS240. Mindray Bio-Medical Electronics Co., Ltd) to estimate the total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), VLDL, TG and non-HDL. Fasting blood glucose was measured with the URIT glucometer (URIT G26®, URIT Medical Electronic, UK).\n\nFor body mass index (BMI) estimation, height was measured to the nearest centimetre without shoes with a stadiometer (Seca 217, 40 Barn Street B5 5QB Birmingham, United Kingdom) and weight was measured to the nearest 0.1 kg, with a bathroom scale (Zhongshan Camry Electronic Co. Ltd, Guangdong, China). BMI was calculated as a ratio of weight (kg) to height squared (m2). This was used to categorize participants as underweight (<18 kg/m2), normal (18–24 kg/m2), overweight (25–29.9 kg/m2) and obese (>30 kg/m2) according to WHO criteria15.\n\nBlood pressure measurements were performed according to the American Heart Association recommendations16. Measurements of blood pressure were performed using an automatic validated device (Omron HEM711DLX, UK) on the superior left limb. The subjects were made to sit with the legs uncrossed and arm supported at the height of the heart with cuff adapted to the size of the arm. Blood pressure was measured as the mean values of duplicate measurements. Grading of hypertension recorded as follows; “normal” when the systolic blood pressure (SBP) was < 120 mmHg and diastolic blood pressure (DBP) was <80 mmHg “pre-hypertension” when SBP = 120–139 or DBP = 80–89 and “hypertension” when SBP = 140–159 or DBP = 90–9917.\n\nAtherogenic indices estimated included AIP (log (TG/HDL), visceral adiposity index (VAI) (VAI=(WC36.5+(1.89×BMI))×(TG0.81)×(1.52HDL)), body adiposity index (BAI) (BAI=100×hipcircumferenceinmheightinm×height−18), non-HDL(TC – HDL) and CRI-I(TC/HDL)18.\n\nData for the study were analyzed using GraphPad prism (6.01) and R statistical software package version 3.0.1. Exploratory analysis was done for descriptive statistical indices such as frequencies, percentages and mean ± standard deviations. Tables were obtained from exploratory analysis. Student’s t-test was performed to compare pre and post-menopausal groups for anthropometrics, lipid profiles and atherogenic indices. Fisher’s exact test or Chi-square test were employed where deem fit to assess the association between proportions of variables in pre and post-menopausal groups. Crude and adjusted odds ratios (aOR) at 95% confidence interval (CI) were evaluated for Fisher’s exact test outcome. Correlation analysis was done using Spearman’s rho moment correlation analysis for lipid profile and atherogenic indices among the study participants. Significance level was determined at P<0.05.\n\n\nResults\n\nA total of 150 pre and post-menopausal women were enrolled into the study with a significantly older post-menopausal group (59.63±7.419, P<0.0001) compared to the pre-menopausal group (32.28±8.820). Also, the post-menopausal group had an elevated SBP (P=0.0003), DBP (P=0.0822), FBS (P=0.0004) as well as a higher BMI compared to the pre-menopausal women (Table 1). Complete demographic information, alongside all other variables measured, are available as Underlying data19.\n\nData presented as mean ± SD.\n\nWC, waist circumference; HC, hip circumference; WC/HC, waist to hip ratio; FBS, fasting blood sugar; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure.\n\nSerum TC, TG and NON-HDL-C were significantly increased in post-menopausal women compared to pre-menopausal women (p<0.0001). In addition, there was an observed significant increase in LDL-C (p<0.0001) and VLDL (p=0.0021) levels in the post-menopausal women. In comparison with pre-menopausal women, atherogenic markers (AIP, VAI, BAI and CRI-I) were significantly elevated in post-menopausal women (p<0.0001) (Table 2).\n\nTC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; VLDL, very low-density lipoprotein; TG, triglycerides; AIP, atherogenic index of plasma; BAI, body adiposity index; CRI-I, Castelli index I.\n\nHigher levels of TC, LDL-C, AIP, BAI and CRI-I, as well as low levels of HDL-C, were all crudely associated with post-menopausal women (p<0.0001). However when the data was adjusted for age and BMI, only elevated levels of TC [aOR=76.58 (95%CI=5.880-2439.396), P=0.0032], LDL-C [aOR=11.76 (95%CI=1.934-90.816), P=0.011], BAI [aOR=41.19 (95%CI=6.7511-321.0692), P=0.0001], CRI-1 [aOR=818.824 (95%CI=51.900-29515.961), P=0.0001] as well as low levels of HDL-C [aOR=11.76 (95%CI=1.934-90.816), P=0.011] were significantly associated with post-menopausal women (Table 3).\n\nNA, odds ratio value not given by R statistical package; aOR, age- and BMI-adjusted odds ratio; CI, confidence interval; TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; VLDL, very low-density lipoprotein; TG, triglycerides; AIP, atherogenic index of plasma; BAI, body adiposity index; CRI-I, Castelli index I.\n\nIn post-menopausal women, we report a positive correlation of TC with all the cardiovascular and atherogenic markers except HDL-C whilst HDL-C on the other hand showed a significant negative correlation with all the atherogenic and cardiovascular markers with the exception of BAI, which wasn’t significant (Figure 1).\n\n\nDiscussion\n\nAlthough CVD is the major cause of death and disability in women, it usually starts about 10 years late in men of the same age. It is also true that CVD are the major cause of mortality in post-menopausal women4. The patterns of dyslipidemia that leads to CVD and its associated complications have been linked with hormonal changes associated with menopause. Estrogen is known to possess both anti-atherogenic and cardioprotective effect by maintaining an acceptable balance between pro/anti-atherogenic and cardiovascular risk markers9. We therefore sought to assess cardiovascular and atherogenic risk among pre and post-menopausal women in the Cape-Coast municipality.\n\nIn comparison to pre-menopausal women, we report elevated levels of TC (p<0.0001), VLDL, TG (p<0.0001), LDL-C (p<0.0001) and NON-HDL cholesterol (p<0.0001) in post-menopausal women. This is in line with earlier findings by Pardhe et al., who also reported significantly increased levels of TG, TC, LDL-C and reduced levels of HDL-C among Nepalese women4. Adverse changes in lipids and lipoprotein independent of age has been linked to mmenopause20. Among all the risk factors for CVD, the major indication suggests an association of estrogen with the observed discrepancies in lipids and lipoproteins12. Earlier studies have reported an increase in the release of free fatty acids into circulation due to high-fat accumulation leading to elevated hepatic triglycerides synthesis21. In addition, a reduction in estrogen after menopause increases plasma lipoprotein lipase (LPL) and hepatic TG lipase activity thereby causing accumulation of plasma LDL-C22. However, HDL-C was significantly decreased in post-menopausal women which is in tandem with the findings of previous studies23,24. Available evidence shows that as HDL-C increases by 0.026 mmol/ml, there is a reduction in risk of cardiovascular diseases, with a 4.7% decrease in mortality rate of CVD25. Changes in plasma lipid is known to partly increase the incidence of cardiovascular disease following menopausal transition20,24.\n\nNewly emerging atherogenic markers such as AIP, VAI, BAI and CRI-I have been used to assess cardiovascular risk and atherogenicity among various disease states including hypertension26, diabetics27 and among HIV patients11. Our results revealed that, markers of atherogenicity including AIP (p<0.0001), VAI (p<0.0001), BAI (p<0.0038) and CRI-I (p<0.0001) were significantly increased in post-menopausal women. This is in line with a study by Nwagha et al., which reported a significantly reduced AIP levels among pre-menopausal women confirming the alteration of lipid profile in menopause26. The use of lipid ratios as prognosticators of cardiovascular risk cannot be overemphasized. In fact, variations in lipid ratios such as CRI-I and CRI-II have been reported to be better predictors of risk reduction in coronary heart disease compared to the absolute lipoproteins or lipids28.\n\nHigher levels of TC, LDL-C, AIP, BAI and CRI-I as well as low levels of HDL-C were all crudely associated with post-menopausal women; however, after correction for age and BMI, AIP was not significantly associated to postmenopausal status with a marked reduction in the significance levels of the other parameters (Table 3). This confirms the significant intersection between cardiovascular risk and aging29. We also report a positive correlation of TC, TG, and VLDL with atherogenic markers AIP, VAI and CRI-1 in post-menopausal women. Upsurge in the levels of TG in isolation has been shown to increase AIP in women than in men but its influence can be neutralized by the levels of HDL30. Others have reported an association of CVD progression with the size of LDL-C and HDL-C, with the lesser size showing great atherogenic potential31. There is indeed a strong relationship between cholesterol etherification rate in HDL plasma (FERHDL) and lipoprotein particle sizes, which is considered as a risk marker of coronary artery diseases. Lately, VAI has demonstrated to be a potent marker of adipose distribution and function indirectly conveying cardiometabolic risk32. Among post-menopausal women, VAI was shown to predict cardiometabolic risk in association with visceral fat33 while positively correlating with peripheral glucose usage during euglycemic hyperinsulinemic clamp32. Among our study population, post-menopausal women showed increased body adiposity with a high percentage of body fat than pre-menopausal counterparts. An easy but effective determination of adiposity is needed to assess the magnitude of cardiovascular disease for the development of suitable management and preventive strategies. Confirmatory studies in different ethnicities have consistently revealed the overestimation and underestimation of adiposity at low and higher body fat percentages, respectively, by BAI34. Therefore, it is important to carefully interpret BAI values along with other anthropometric and cardiovascular markers.\n\nOur results also indicated there was a significant increase in BMI, blood pressure and fasting blood sugar in post-menopausal women in contrast with pre-menopausal women. The increase may be attributed to the reduction in the production of estrogen, which may be associated with amplified cardiovascular risk in post-menopausal women. Surgical or naturally induced menopause increases the risk of CVD35 and the changeover may be associated with changes in body composition, with a considerable increase in the waist-to-hip ratio during menopause coupled to a possible increase in BMI36. Research has demonstrated the upsurge in the release of nitric oxide and the production of prostacyclin within the arterial endothelial cells by estrogen, culminating into the induction of a vasodilatory effect leading to a drop in BP37. Furthermore, estrogen again reduces the synthesis of thromboxane A2 by platelets with vasoconstriction properties38. Hence the absence or a reduction in the estrogen levels during menopause may increase blood pressure due to an increase in peripheral resistance. Also, when estrogen production is low, there is reduced stimulation of the liver to synthesize renin. This is a rate-limiting step in the renin-angiotensin-aldosterone system leading to vasoconstriction with subsequent increase in blood pressure39. On the other hand, urbanization, affluence, changing dietary habits and sedentary lifestyles can also be implicated for the above findings40. Even though a significant association of increased cardiovascular risk with post-menopausal status was found in this study, our findings may be limited by the smaller sample size used and hence a larger sample size would have provided more stability to our conclusions.\n\n\nConclusion\n\nMenopause could lead to changes in lipid profile in the direction of atherogenicity and increase the risk of CVDs. Atherogenic markers such as AIP, VAI, BAI, and CR can serve as potential biomarkers for predicting CVD and can be used together with other markers.\n\n\nData availability\n\nZenodo: Assessment of cardiovascular risk in post-menopausal women in Ghana. http://doi.org/10.5281/zenodo.322891719.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSherwin B: Menopause: Myths and realities. Psychological aspects of women's health care, the interface between psychiatry and obstetrics and gynecology. 1993; 227–248. Reference Source\n\nTrishala A, Priya VV, G R: Comparative assessment of lipid profile in pre- and post-menopausal women in Tuticorin district –a pilot study. Int J Pharm Bio Sci. 2016; 7(3): 1109–12. Reference Source\n\nFernandez ML, Murillo AG: Postmenopausal Women Have Higher HDL and Decreased Incidence of Low HDL than Premenopausal Women with Metabolic Syndrome. Healthcare (Basel). 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Diabetes Metab Res Rev. 2009; 25(8): 733–739. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Obesity: preventing and managing the global epidemic. World Health Organization. 2000. Reference Source\n\nKirkendall WM, Burton AC, Epstein FH, et al.: Recommendations for human blood pressure determination by sphygmomanometers. Circulation. 1967; 36(6): 980–988. PubMed Abstract \| Publisher Full Text\n\nVerdecchia P, Angeli F: [The Seventh Report of the Joint National Committee on the Prevention, Detection, Evaluation and Treatment of High Blood Pressure: the weapons are ready]. Rev Esp Cardiol. 2003; 56(9): 843–847. PubMed Abstract\n\nNansseu JR, Moor VJ, Nouaga ME, et al.: Atherogenic index of plasma and risk of cardiovascular disease among Cameroonian postmenopausal women. Lipids Health Dis. 2016; 15(1): 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOpoku YK, Afrifa J: Assessment of cardiovascular risk in post-menopausal women in Ghana [Data set]. Zenodo. 2019.\n\nStevenson JC, Crook D, Godsland IF: Influence of age and menopause on serum lipids and lipoproteins in healthy women. Atherosclerosis. 1993; 98(1): 83–90. PubMed Abstract \| Publisher Full Text\n\nTankó LB, Bagger YZ, Qin G, et al.: Enlarged waist combined with elevated triglycerides is a strong predictor of accelerated atherogenesis and related cardiovascular mortality in postmenopausal women. Circulation. 2005; 111(15): 1883–1890. PubMed Abstract \| Publisher Full Text\n\nWakatsuki A, Okatani Y, Ikenoue N, et al.: Effect of lower dose of oral conjugated equine estrogen on size and oxidative susceptibility of low-density lipoprotein particles in postmenopausal women. Circulation. 2003; 108(7): 808–813. PubMed Abstract \| Publisher Full Text\n\nJensen J, Nilas L, Christiansen C: Influence of menopause on serum lipids and lipoproteins. Maturitas. 1990; 12(4): 321–331. PubMed Abstract \| Publisher Full Text\n\nMatthews KA, Meilahn E, Kuller LH, et al.: Menopause and risk factors for coronary heart disease. N Engl J Med. 1989; 321(10): 641–646. PubMed Abstract \| Publisher Full Text\n\nOkonofua FE, Lawal A, Bamgbose JK: Features of menopause and menopausal age in Nigerian women. Int J Gynaecol Obstet. 1990; 31(4): 341–345. PubMed Abstract \| Publisher Full Text\n\nNwagha UI, Ikekpeazu EJ, Ejezie FE, et al.: Atherogenic index of plasma as useful predictor of cardiovascular risk among postmenopausal women in Enugu, Nigeria. Afr Health Sci. 2010; 10(3): 248–52. PubMed Abstract \| Free Full Text\n\nOkpa HO, Enang OE, Effa EE, et al.: Comparative analysis of atherogenic index of plasma and its relationship with cardiovascular risk among patients with diabetes mellitus and concurrent diabetes mellitus with hypertension attending endocrinology clinic in a tertiary hospital south-south Nigeria. atherosclerosis. 2015; 13(15): 16. Reference Source\n\nMillán J, Pintó X, Muñoz A, et al.: Lipoprotein ratios: Physiological significance and clinical usefulness in cardiovascular prevention. Vasc Health Risk Manag. 2009; 5: 757–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNorth BJ, Sinclair DA: The intersection between aging and cardiovascular disease. Circ Res. 2012; 110(8): 1097–1108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStensvold I, Tverdal A, Urdal P, et al.: Non-fasting serum triglyceride concentration and mortality from coronary heart disease and any cause in middle aged Norwegian women. BMJ. 1993; 307(6915): 1318–1322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrexel H, Amann FW, Rentsch K, et al.: Relation of the level of high-density lipoprotein subfractions to the presence and extent of coronary artery disease. Am J Cardiol. 1992; 70(4): 436–440. PubMed Abstract \| Publisher Full Text\n\nAmato MC, Giordano C: Visceral adiposity index: an indicator of adipose tissue dysfunction. Int J Endocrinol. 2014; 2014: 730827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmato MC, Giordano C, Pitrone M, et al.: Cut-off points of the visceral adiposity index (VAI) identifying a visceral adipose dysfunction associated with cardiometabolic risk in a Caucasian Sicilian population. Lipids Health Dis. 2011; 10(1): 183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamírez-Vélez R, Correa-Bautista JE, González-Ruíz K, et al.: Body Adiposity Index Performance in Estimating Body Fat Percentage in Colombian College Students: Findings from the FUPRECOL-Adults Study. Nutrients. 2017; 9(1): pii: E40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar S, Shah C, Oommen E: Study of cardiovascular risk factors In pre and postmenopausal women. Int J Pharma Sci Res. 2012; 3(12): 560–570. Reference Source\n\nWalton C, Lees B, Crook D, et al.: Body fat distribution, rather than overall adiposity, influences serum lipids and lipoproteins in healthy men independently of age. Am J Med. 1995; 99(5): 459–464. PubMed Abstract \| Publisher Full Text\n\nTostes RC, Nigro D, Fortes ZB, et al.: Effects of estrogen on the vascular system. Braz J Med Biol Res. 2003; 36(9): 1143–1158. PubMed Abstract \| Publisher Full Text\n\nCheang A, Sitruk-Ware R, Samsioe G: Transdermal oestradiol and cardiovascular risk factors. Br J Obstet Gynaecol. 1994; 101(7): 571–581. PubMed Abstract \| Publisher Full Text\n\nAtlas SA: The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. J Manag Care Pharm. 2007; 13(8 Supp B): 9–20. PubMed Abstract \| Publisher Full Text\n\nGarg B, Yadav N, Vardhan H, et al.: Asymptomatic obese hypertensives and need of routine echocardiography for left ventricular mass assessment and treatment. J Clin Diagn Res. 2013; 7(8): 1599–603. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "49750", "date": "24 Jun 2019", "name": "Bashu Dev Pardhe", "expertise": [ "Reviewer Expertise Metabolic diseases", "Enzymology (Cytochrome P450)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 fact relies on that cardiovascular diseases (CVDs) are more prevalent in women after menopause; the researchers compare the lipid profile and atherogenic index in pre- and post-menopausal women. They included a total of 150 women and estimated BP, FBG and lipid profile and finally, they determined the atherogenic markers like BMI, AIP, VAI, BAI, and CRI-I for the interpretation. They found a significant rise in lipid profiles except for HDL-C in post-menopausal women compared with pre-menopausal women. Mean AIP, VAI, BAI, and CRI-I (p<0.0001) were significantly increased in post-menopausal women compared to pre-menopausal women. Higher levels of BAI and CRI-I were significantly associated with post-menopausal women after even adjusted for age and BMI factors. At the same time, they reported a positive correlation of TC, TG, VLDL-C and non-HDL-C with atherogenic markers AIP, VAI and CRI-I in post-menopausal women. With these findings, they conclude that postmenopausal women have an adverse change in lipid parameters with an increased atherogenic index and they are more prone to have cardiovascular diseases.\n\nComments:\nI found the manuscript interesting from an epidemiological point of view, which has the value to describe the lipid profile and atherogenicity in a group of pre and postmenopausal women from Ghana.\n\nThe age range for postmenopausal women was 40-55 years and for pre-menopausal was 30-40 years in the method section. In the result section (table 1), the mean age of post-menopausal women was reported to be 59.63±7.42. How is this possible?\n\nSocio-demographic factors like lifestyle interventions, education levels, social status, ethnicity, etc. may also influence the findings of this study. It will be useful (and interesting) to have a table with a more complete description of the women included in the study.\n\nThe author mentioned, “We, therefore, sought to assess cardiovascular and atherogenic risk among pre and post-menopausal women in the Cape-Coast municipality.” The selection criteria and sample size do not reflect this statement.\n\nThe selection of the study population is not clearly defined. They approach for the convenient sampling, it is better to define inclusion and exclusion criteria for both pre and post-menopausal women separately. They recruit healthy women attending the hospital. The question remains why healthy women do attend the hospital. Were they completely healthy or apparently healthy?\n\nWide range of age of study population required larger sample size. Even author mention that the sample size limit this cross-sectional study, this relative smaller sample size may sometimes miss-lead the conclusion.\n\nI recommend including significance of atherogenic index (AIP, VAI, and BAI) in introduction section because this study focused on the investigation of these parameters.\n\nTable 1:\nThe average weight of post-menopausal women (6.21±8.82 kg)? I recommend using the same SI unit (cm or m) for height, WC and HC. I recommend using the maximum 3 digits after decimal and uniform for all variables (all tables and figure). Mention the criteria to define pre-diabetes in study population at least in the method section.\n\nIt is better to include the abbreviation section in the manuscript. Please use cardiovascular diseases (CVDs), very low-density lipoprotein cholesterol (VLDL-C).\n\nTitle of the tables should not be end with a full stop.\n\nEven though the findings are more interesting, interpretation and discussion part is poor. The author reported the association of the atherogenic index with menopausal status with respect to adjusted for age and BMI. The discussion part is poor to justify this analysis.\n\nThere are some typo errors especially in abbreviations (mainly on abstract) and grammatical errors in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": [] }, { "id": "49748", "date": "04 Nov 2019", "name": "Christian Obirikorang", "expertise": [ "Reviewer Expertise Chemical 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\nOverview and General Recommendations:\nThank you for the opportunity to review this manuscript which highlights the need for CVD risk assessment among postmenopausal women. Fat mass accumulation is one of the significant changes that occur with advancing age and its peaks around the menopausal, and it is associated with metabolic and haemodynamic derangement. The research interests of the paper is that it adds to already existing knowledge on menopause and CVD risk. Despite the fact that menopausal transition is associated with physiological changes which affects the health status of women, little data exist in Ghana. Thus highlighting the relevance of the paper in context.\nI found the paper to be overall well written and much of it to be well described. I felt confident that the authors executed careful and thorough processing of methods to generate the data. The design of the study combined with data processing strategy makes the data set seem quite useful for the purpose. However, there are few concerns which a minor revision is warranted.\nFirst, the authors placed too much emphasis on lipids and anthropometry given little attention to the constellation of metabolic abnormalities (also called metabolic syndrome) and hypertension which are potent indicators of CVD. Lipids abnormalities or atherogenic indices alone does not explain 100% of CVD risk prevalence among postmenopausal women. Dyslipidaemia increases the risk of coronary artery disease among and lipid peroxidation promote atherosclerosis. Thus making the single emphasis on dyslipidemia relevant. However, for general CDD risk, I strongly recommend that the authors should include MetS and hypertension, which will give the relevance of the study in context.\nMinor Revisions:\nAt the abstract section, the authors should correct “comparative cross-section” and change it to “comparative cross-sectional study”.\nAt the results section, the mean blood pressure were within the recommended range for both groups. Thus, the first sentence should be revise: Also postmenopausal group had elevated BP should be change to “a higher BP levels were observed among postmenopausal women…… or something similar.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-845
https://f1000research.com/articles/8-844/v1	11 Jun 19	{ "type": "Data Note", "title": "Mass spectrometry data of diabetic rat sperm proteome treated with Gynura procumbens aqueous extract", "authors": [ "Khaidatul Akmar Kamaruzaman", "Wan Mohd Aizat", "Mahanem Mat Noor", "Khaidatul Akmar Kamaruzaman", "Wan Mohd Aizat" ], "abstract": "Diabetes mellitus has a deleterious effect on the male reproductive system, especially on sperm quality and spermatogenesis. Gynura procumbens (G. procumbens) is a traditional herb known for its ability to improve the fertility of diabetes-induced male rats. This study was designed to identify the differential expression of sperm proteins after treatment with G. procumbens aqueous extract on diabetes-induced male rats. The sperm proteome was profiled using label-free shotgun proteomics analysis. Sprague Dawley rats used in this study were divided randomly into four groups. One group was a normal control group (healthy rats), while the three other groups were induced with 50 mg/kg bodyweight (BW) of streptozotocin (STZ) to emulate the diabetic condition. The diabetic rats were divided into negative control (non-treated diabetic), metformin-treated (positive control) and G. procumbens aqueous extract-treated (450 mg/kg BW) groups. Oral treatments were administered for 14 consecutive days before the rats were euthanized. Total sperm protein samples were extracted from the caudal epididymis and run through SDS-PAGE. Later, samples were digested using trypsin before liquid chromatography-tandem mass spectrometry (Thermo Orbitrap Fusion) analysis. The acquired data were processed using MaxQuant and Perseus software. The mass spectrometry proteomics data is available through ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PXD011373.", "keywords": [ "Gynura procumbens", "diabetes mellitus", "fertility", "proteomic", "Sambung nyawa" ], "content": "Introduction\n\nDiabetes mellitus is a metabolic disorder that is often associated with male infertility, causing disruption to spermatogenesis, testicular impairment, and erectile and ejaculation dysfunction1. Disruption to spermatogenesis and testicular impairment in diabetic rats has been reported to cause low sperm quality2. Current medicine, such as metformin and other oral anti-hyperglycaemic drugs, claimed to significantly reduced blood glucose level; however, they could not improve the fertility problems associated with diabetes3. Medicinal plants or herbs have been long used in traditional practice and can be considered as alternative treatments for some diseases. Gynura procumbens is one of the herbs that has shown potential for treating various diseases such as diabetes mellitus, cancer and, recently, fertility. A previous study demonstrated that G. procumbens had potential as anti-hyperglycaemic agent by lowering the blood glucose level of diabetes-induced male rats4. It is claimed that G. procumbens imitates the mechanism of action of insulin, increasing the glucose uptake into the skeletal muscle5. Additionally, G. procumbens extract has the ability to improve the fertility of diabetes-induced male rats3,6. To further elucidate the effect of G. procumbens aqueous extract (GPAE) on sperm quality, a proteomic analysis was performed to identify and quantify the differential expression of total sperm proteins after treatment with G. procumbens extract.\n\n\nMaterials and methods\n\nThis research has obtained ethical approval from the Animal Ethics Committee of Faculty of Medicine, Universiti Kebangsaan Malaysia (FST/2013/MAHANEM/31-JAN./492-FEB.-2013-FEB.-2015). All efforts were made to ameliorate harm to animals, achieved by inducing to imitate diabetes conditions with streptozotocin (STZ) intravenously at a dose of 50 mg/kg bodyweight (BW) in all groups except the control normal group, which was left uninduced.\n\nA total of 28 male Sprague Dawley rats aged eight weeks (120–200 g) were used in this study. All rats were proven fertile and provided by the Animal House of Universiti Kebangsaan Malaysia. All animals were acclimatized for seven days, wherein their food pellet (Rat Chow, Barastoc, Ridley, Australia) and drink intakes were given ad libitum. The rats were kept in PVC cages at controlled room temperature with a 12-hour light/12-hour dark cycle.\n\nG. procumbens were harvested from Universiti Kebangsaan Malaysia glass house (2.9300°N, 101.7774°E). The G. procumbens aqueous extract was prepared as described previously3. Briefly, the leaves part of G. procumbens were harvested and dried in the oven for 72 hours at 48°C. The dried leaves then were ground to dust and extracted for three hours at 60°C. The extract produced was in liquid form, later sent for freeze-drying and kept in 4°C for freshness.\n\nExperimental methods and procedures were detailed as previously7. Briefly, male Sprague-Dawley rats were used in this study, which were randomly hand-picked and divided into four groups, with seven rats in each group. Randomization was performed in choosing the rats to avoid bias in the experiment. One group served as a normal untreated group (N), while the three other groups were induced with 50 mg/kg BW STZ intravenously for type 1 diabetes induction. The blood glucose level of each rats was measured after 72 hours of STZ induction. Rats with blood glucose level at 13 mmol/L and more were considered as diabetic and used in this study. These diabetic rats served as (i) diabetic-untreated (negative control), (ii) diabetic-metformin treated (positive control) (500 mg/kg BW) and (iii) diabetic-GPAE treated (450 mg/kg BW) groups. Treatment was given via oral gavage for 14 consecutive days after seven days of STZ induction at the Animal House, Universiti Kebangsaan Malaysia. On the 15th day, all rats were euthanized by inhalation of diethyl ether for sperm protein analysis.\n\nSperm protein extraction was performed as described previously8. Briefly, sperm samples from each rat were collected separately from the caudal epididymis and minced in Biggers-Whitten-Whittingham medium (91.06 mM Sodium chloride, 4.78 mM Potassium chloride, 2.0 mM Calcium chloride, 1.17 mM potassium phosphate, 2.44 mM Magnesium sulphate, Sodium hydrogen carbonate, 0.25 mM Sodium pyruvate, 21.55 mM Sodium lactate, 5.55 mM glucose, and bovine serum albumin)9. Briefly, the samples were incubated in a 5% CO2 incubator for 30 minutes at 37°C, to allow the sperm to swim up. Sperm samples were then centrifuged at 4000 rpm for 15 minutes and the supernatant was removed. The pellet was mixed with lysis buffer (7 M Urea, 2 M Thiourea, 4% 3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), 0.8% Immobilized pH Gradient (IPG) buffer, 1 mM phenylmethane sulfonyl fluoride (PMSF)). The samples were then centrifuged at 15,000 rpm for 20 minutes at 4°C. The supernatant was collected, added to 60 mM dithiothreitol (DTT) and kept at 80°C until used. The concentration of sperm proteins was determined using the Bradford assay10.\n\nA total of 100 µg of the sperm protein samples from each group were used for one-dimensional SDS-PAGE. Protein samples were loaded onto 12.5% SDS-PAGE gels and run for eight minutes or until all the proteins stacked up in the resolving gel. The gel was then cut for trypsin digestion using the in-gel trypsin digestion method11. For this purpose, excised protein bands were treated with DTT for one hour at 57 °C to reduce the disulphide bridges, followed by Iodoacetamide for another hour at room temperature in the dark for alkylation purposes, as previously described11. Gels were rehydrated with 50 mM ammonium bicarbonate and dehydrated with acetonitrile for three times, respectively. Enzymatic digestion was performed by incubating samples with trypsin for 30 minutes at 4 °C in a 1: 50 (w/w) ratio. The digestion products were then incubated with 50 mM ammonium bicarbonate for overnight at 37 °C. Digested peptides were sent for liquid chromatography with tandem mass spectrometry (LC-MS/MS) for protein identification and quantification.\n\nLC-MS/MS analysis was performed as described in Kamaruzaman et al.7. Briefly, the peptide samples were analysed using a liquid chromatography (LC) system (UltiMate 3000 RSLCnano, Dionex) coupled to a Linear Trap Quadropole (LTQ) Orbitrap Fusion mass spectrometer (Thermo Fisher, Bremen, Germany). A total of 1.0 µL of digested samples from each group were injected into a reverse phase column (15 cm × 75 µm internal diameter, particle size of 2 µm, 100 Å, C18 PepMap column) and eluted at a flow rate of 300 nL/min. The elution mobile phase composition was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The eluted peptides were separated in 5–40% solvent B for 91 minutes, 95% solvent B for 2 minutes, 95% solvent B for 6 minutes and back to 5% solvent B for 2 minutes. Data were acquired in data dependent mode. Full scan spectra in the range of 310–1800 m/z were acquired. The automatic gain control (AGC) was targeted at 4.0 × 105 with a maximum injection time of 50 ms and 3 seconds in top speed mode, where precursors were selected with a maximum cycle time of 3 seconds. Precursors with an assigned monoisotopic m/z and a charge between 2 and 7 were further analysed. All precursors were filtered using a 20 second dynamic exclusion window with an intensity threshold of 5000. The MS/MS spectra analyses were performed using rapid scan rate and collision-induced dissociation (CID), with normalized collision energy (NCE) set to 30% and high energy collision-induced dissociation (HCD) at 28%, a 1.6 m/z isolation window, AGC targeted at 1.0 × 102 and a maximum injection time of 250 ms.\n\nData processing was conducted according to previous studies12,13. Raw files for each biological replicate were analysed together using the MaxQuant software (version 1.5.3.30)14. The derived peak list was searched using the Andromeda search algorithm embedded in the MaxQuant workflow. The protein sequence database used for protein identification analysis was the Rattus norvegicus database, obtained from Uniprot database (proteome ID: UP000002494, accessed on February 2016). The MaxQuant parameters were set as enzyme trypsin and allowed up to two miss cleavages. The peptide length was set minimally up to seven amino acids. The spectra search included carbamidomethylation of cysteine as a fixed modification and oxidation of methionine was set as a variable modification. Since no labelling was performed, multiplicity was set to one. Peptide spectrum match (PSM) and protein identification were filtered using a target-decoy approach with a false discovery rate (FDR) of 1 %. The second peptide feature was enabled. The label-free quantification (LFQ) of protein was done using the MaxLFQ algorithm integrated in MaxQuant. Other MaxQuant settings were set as default. All resulting information was reported in the “proteinGroups” output file (proteinGroups.txt in the combined file), containing the full list of identified and quantified proteins. This file has been deposited to the ProteomeXchange Consortium15 via the PRIDE partner repository with the dataset identifier PXD011373.\n\nThe data were further analysed for protein quantification using Perseus software (version 1.5.4.1)16. The hits to the reverse database, contaminants and proteins identified with modified peptides were eliminated. Then, the LFQ intensity ratios were transformed by log2. Missing values were imputed by drawing random numbers from a normal distribution to stimulate signals from low abundance proteins, using the default parameters described previously12. The value of log2 (LFQ ratios) for each sample were averaged and statistically analysed as per Kamaruzaman et al.7. In short, data were displayed as mean ± standard error of mean. The statistical analysis for protein LFQ intensities were compared using Perseus software (version 1.5.4.1) (one-way ANOVA, p < 0.05).\n\n\nDataset\n\nThe dataset is composed of raw and processed LC-MS/MS data for the proteome profiling of rat sperm. Samples were collected from the caudal epididymis of four different groups of rats; normal-untreated, diabetic-untreated (negative control), diabetic-metformin treated (positive control) and lastly, diabetic-GPAE treated. LC-MS/MS, followed by MaxQuant and Perseus analyses, were carried out to identify and quantify the total sperm proteins (Table 1).\n\nProtein identification revealed 473 proteins, and using a stringent search in MaxQuant software (including filtering contaminants and hits to decoy database), a total of 88 proteins were found in all groups. Identified proteins were later quantified using MaxQuant and Perseus software. This information revealed the effect of G. procumbens on the male diabetic rat reproductive system, specifically on the sperm proteins that are involved in fertilization.\n\n\nSummary\n\nThe elucidation of sperm proteins using high-throughput proteomics techniques is still rather limited, especially in complementary medicine studies. Most of the previous studies17,18, especially those involving herbal extract treatments on murine model organisms, mainly utilized a 2D gel-based approach, which is far less sensitive than shotgun proteomics19. Most of these studies identified only around 200–300 proteins, which may not profile the entirety of the sperm proteome. This shortcoming may be due to the low throughput of the gel technique, as well as the inability to visualize low-abundance proteins16.\n\nOn the other hand, shotgun proteomics mainly relies on the acquisition of peptide identities from digested total protein samples using an LC-MS/MS approach. In this study, 2411 peptides were found, which corresponds to 473 proteins. The high number of identified proteins suggests the applicability and sensitivity of shotgun proteomics, as compared to the more conventional 2D gel approach. Hence, shotgun proteomics, particularly using LC-MS/MS Orbitrap, can be considered as an effective strategy for producing a comprehensive proteome profiling of rat sperm, especially regarding GPAE treatment. Additionally, this dataset can be used as a primary guide for the characterisation of protein biomarkers for sperm quality and fertility in male rats.\n\n\nData availability\n\nDataset on ProteomeXchange, Accession number PXD01133: https://identifiers.org/px/PXD011373\n\nRattus norvegicus reference proteome was obtained from Uniprot, Proteome ID UP000002494: https://www.uniprot.org/proteomes/UP000002494.", "appendix": "Grant information\n\nThis research has been funded by Economic Transformation Programme Research Fund (ETP-2013-080) and a Research University Grant (GUP-2016-056).\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\nAlves MG, Rato L, Carvalho RA, et al.: Hormonal control of Sertoli cell metabolism regulates spermatogenesis. Cell Mol Life Sci. 2013; 70(5): 777–793. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoor MM, Nani RMR: Anti-hyperglycemic effect of Gynura procumbens methanolic extract on fertility and libido of induced diabetic male rats. Sains Malays. 2012; 41(12): 1549–1556. Reference Source\n\nKamaruzaman KA, Aizat WM, Mat Noor M: Gynura procumbens Improved Fertility of Diabetic Rats: Preliminary Study of Sperm Proteomic. Evid Based Complement Alternat Med. 2018; 2018: 9201539. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYunianto I, Bashah NAK, Noor MM: Antifertility properties of Centella asiatica ethanolic extract as a contraceptive agent: Preliminary study of sperm proteomic. Asian Pac J Reprod. 2017; 6(5): 212–216. Publisher Full Text\n\nBiggers JD, Whitten WK, Whittingham D: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology. Daniel JC. Freeman (Ed), San Francisco, 1971; 86–116. Reference Source\n\nBradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72(1–2): 248–254. PubMed Abstract \| Publisher Full Text\n\nRamm SA, McDonald L, Hurst JL, et al.: Comparative proteomics reveals evidence for evolutionary diversification of rodent seminal fluid and its functional significance in sperm competition. Mol Biol Evol. 2009; 26(1): 189–198. PubMed Abstract \| Publisher Full Text\n\nHamezah HS, Durani LW, Yanagisawa D, et al.: Proteome profiling in the hippocampus, medial prefrontal cortex, and striatum of aging rat. Exp Gerontol. 2018; 111: 53–64. PubMed Abstract \| Publisher Full Text\n\nMostafa M, Jahar NA, Azizan KA, et al.: Proteomic analysis of stored core oil palm trunk (COPT) sap identifying proteins related to stress, disease resistance and differential gene/protein expression. Sains Malays. 2018; 47(6): 1259–1268. Publisher Full Text\n\nCox J, Mann M: MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008; 26(12): 1367–1372. PubMed Abstract \| Publisher Full Text\n\nVizcaíno JA, Deutsch EW, Wang R, et al.: ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nature Biotechnol. 2014; 30(3): 223–226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTyanova S, Temu T, Sinitcyn P, et al.: The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016; 13(9): 731–740. PubMed Abstract \| Publisher Full Text\n\nYunianto I, Bashah NAK, Noor MM: Antifertility properties of Centella asiatica ethanolic extract as a contraceptive agent: Preliminary study of sperm proteomic. Asian Pac J Reprod. 2017; 6(5): 212–216. Publisher Full Text\n\nLuthfi MJ, Kamalrudin A, Noor MM: Effects of Lunasia amara blanco (Sanrego) on male fertility: A preliminary study on sperm proteomic analysis. Journal of Applied Pharmaceutical Science. 2017; 7(8): 85–92. Publisher Full Text\n\nIovinella I, Caputo B, Michelucci E, et al.: Candidate biomarkers for mosquito age-grading identified by label-free quantitative analysis of protein expression in Aedes albopictus females. J Proteomics. 2015; 128: 272–279. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59564", "date": "21 Feb 2020", "name": "Yun Shin Sew", "expertise": [ "Reviewer Expertise Proteomics", "Transcriptomics", "Metabolomics", "Microbiome." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presented proteomics datasets on the sperm quality of diabetic male rats treated with Gynura procumbens extract. The study was well planned, executed and the article is well drafted. Indeed, the authors addressed the effectiveness of this G. procumbens medicinal plant in treating diabetes mellitus and infertility in a previously published article by Kamaruzaman et al. (2018)1. While in this article, the authors detailed out the experimental design and methodology for the preparation of proteomic data and how the data were analysed using MaxQuant and Perseus analysis softwares. However, there are a number of issues which need to be clarified and additional information are necessary in order to improve this article:\nGiven that the raw and processed data have been deposited in Proteome Xchange database (PXD011373), however the link for the ‘Dataset FTP location’ seems to be not working. By trying another link given under ‘PRIDE project URI’, the file with name ‘combine.rar’ was downloadable but failed to be opened. I would suggest the authors to check again on the data accessibility issue and fix any possible error in order to share those information with readers.\n\nFor the section ‘Dataset’, additional information are needed particularly related to the processed data. For example, the number of animal sample per group used for generating the processed data, how was the raw data of biological replicates in each group combined for analysis? Was there a normalization step across datasets for each group performed? What were the parameters used for stringent search in MaxQuant and Perseus analysis softwares, including thresholds and cut off values set for identifying a total of 88 proteins in all groups?\n\nThere were three different dosages of G. procumbens aqueous extract (150, 300 and 450 mg/kg) used for treatment referring to Kamaruzaman et al.(2018)1, however this article presented and deposited dataset for only one treatment 450 mg/kg. Perhaps the authors can state somewhere in the article (i.e. experimental design section) regarding the rationale of choosing this particular treatment dataset?\n\nUnder the section ‘Sample collection and preparation’ there was a typo observed in the second last sentence where the samples kept at 80°C until used, it should be -80°C instead.\nIn short, I would recommend this article to be indexed with minor revisions as the above.\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? Partly", "responses": [] }, { "id": "70356", "date": "14 Sep 2020", "name": "María Eugenia Cabrillana", "expertise": [ "Reviewer Expertise Male reproduction." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 entitled \"Mass spectrometry data of diabetic rat sperm proteome treated with Gynura procumbens aqueous extract\", was clearly written.\n\nThe datasets were not clearly presented and the format was not accessible.\n\nPerhaps a table showing the difference or not, in the proteins detected by mass spectrometry, between the control and the experimental groups could improve this article. It is not easy for the reader to look for the results in the PRIDE database.\n\nIn the summary, no reference was made about the effect of the Gynura procumbens aqueous extract, on the proteins analyzed in all groups. Therefore it is not possible to understand whether this technique is useful for detecting differences between treatments.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/8-844
https://f1000research.com/articles/7-30/v1	09 Jan 18	{ "type": "Research Article", "title": "Does evidence support the high expectations placed in precision medicine? A bibliographic review", "authors": [ "Jordi Cortés", "José Antonio González", "María Nuncia Medina", "Markus Vogler", "Marta Vilaró", "Matt Elmore", "Stephen John Senn", "Michael Campbell", "Erik Cobo", "José Antonio González", "María Nuncia Medina", "Markus Vogler", "Marta Vilaró", "Matt Elmore", "Stephen John Senn", "Michael Campbell", "Erik Cobo" ], "abstract": "Background: Precision medicine is the Holy Grail of interventions that are tailored to a patient’s individual characteristics. However, the conventional design of randomized trials assumes that each individual benefits by the same amount. Methods: We reviewed parallel trials with quantitative outcomes published in 2004, 2007, 2010 and 2013. We collected baseline and final standard deviations of the main outcome. We assessed homoscedasticity by comparing the outcome variability between treated and control arms. Results: The review provided 208 articles with enough information to conduct the analysis. At the end of the study, 113 (54%, 95% CI 47 to 61%) papers find less variability in the treated arm. The adjusted point estimate of the mean ratio (treated to control group) of the outcome variances is 0.89 (95% CI 0.81 to 0.97). Conclusions: Some variance inflation was observed in just 1 out of 6 interventions, suggesting the need for further eligibility criteria to tailor precision medicine. Surprisingly, the variance was more often smaller in the intervention group, suggesting, if anything, a reduced role for precision medicine. Homoscedasticity is a useful tool for assessing whether or not the premise of constant effect is reasonable.", "keywords": [ "Constant Effect", "Precision medicine", "Homoscedasticity", "Clinical Trial", "Variability", "Standard deviation", "Review" ], "content": "Introduction\n\nThe idea behind precision medicine is to develop prevention and treatment strategies that take into account individual characteristics. With this strong endorsement “The prospect of applying this concept broadly has been dramatically improved by recent developments in large-scale biologic databases (such as the human genome sequence), powerful methods for characterizing patients (such as proteomics, metabolomics, genomics, diverse cellular assays, and mobile health technology), and computational tools for analyzing large sets of data.”, US President Obama launched the Precision Medicine initiative in 2015 to capitalize on these developments1,2. However, we are not convinced that this is a sensible idea.\n\nVariability of a clinical trial outcome measure should interest researchers because it conveys important information about whether there is a need for precision medicine. Does variance come only from unpredictable and ineluctable sources of patient variability? Or should it also be attributed to a different treatment effect that requires more precise prescription rules3–5? Researchers assess treatment effect modifications (“interactions”) among subgroups based on relevant variables. The main problem with that methodology is that, by the usual standards of classical phase III trial, the stratification factors must be known in advance and be measurable. This in turn implies that when new variables are discovered and introduced into the causal path, new clinical trials are needed. Fortunately, one observable consequence of a constant effect is that the treatment will not affect variability, and therefore the outcome variances in both arms should be equal (“homoscedasticity”). If this homoscedasticity holds, there is no need to repeat the clinical trial once a new possible effect modifier becomes measurable.\n\nNevertheless, the fundamental problem of causal inference is that for each patient in a parallel group trial, we can know the response for only one of the interventions. That is, we observe their response to either the new Treatment or to the Control, but not both. By experimentally controlling unknown confounders through randomization, a clinical trial may estimate the averaged causal effect. In order to translate this population estimate into effects for individual patients, additional assumptions are needed. We try to elucidate whether the comparison of observed variances may shed some light on the non-observable individual treatment effect. See examples and references that illustrate in their interpretation in Figure 16–15.\n\nBecause of the random allocation to one of two treatment arms, we will only observe one of the two potential outcomes for each patient: either under T or under C. Fully saturated colors represent observed Systolic Blood Pressure (SBP) values, and transparent squares represent missing potential values. The line slope indicates the individual non-observable effect for each patient. Densities are the potential distributions of the outcome in each group: As both random samples come from the same target population, the average causal effect is estimable without bias. Panel A shows the potential outcome values that we could obtain if there were not any treatment effect; as the intervention has no effect at all, both groups have the same distribution (i.e., mean and variance). Panel B shows the scenario of a constant effect, meaning that the intervention lowers the SBP by a single value in every patient, implying the same variability in both arms. For instance, the study from Duran-Cantolla et al.6 compared the 24-hour systolic blood pressure among 340 patients randomized to either Continuous Positive Airway Pressure (CPAP) or sham–CPAP, and it showed a greater decrease of 2.1 mmHg (95% CI from 0.4 to 3.7) in the intervention group compared to the control group. Furthermore, baseline standard deviations (SDs) were 12 and 11; and final SDs were 13 for both groups. Therefore, their results fully agree with the trial design’s assumption of a constant effect (scenario B) and nothing contradicts the inference that each patient exhibits a constant reduction of 2.1mmHg, although the uncertainty of random allocation makes the results compatible with a constant effect that lies anywhere between 0.4 and 3.7. Panel C represents a situation with 2 different effects in 2 subpopulations (“treatment by subgroup interaction”). Although the effects are identical within them, the observable distribution in the treated arm would have higher variability. Here, we need to find finer eligibility criteria for classifying patients in those subpopulations so that a constant effect could be assumed again. In Panel D, the treatment has a variable effect in each patient, resulting also in greater variability within the treated arm but without any subgroup sharing a common effect, and results are poorly predictive about the effects on future patients. In the study by Kojima et al.7, the primary outcome measure was the 3-hour postprandial area under the curve of apolipoprotein B48, with outcome SDs being 0.78 and 0.16 in the treated and reference arms, respectively, thus showing a variance ratio of 23.77. This is compatible with different treatment effects that could need further refinements through precision medicine, since a greater variance in the treated arm indicates that “the interpretation of the main treatment effect is controversial”8. In that case, guidelines for treating new patients should be based either on additional eligibility criteria (“precision medicine”, panel C) or on n-of-1 trials (“individualized medicine”, panel D)9–13. This “treatment by patient interaction” was already highlighted by W. S. Gosset in the data of his 1908 paper proposing the Student t-distribution14. Alternatively, interactions can result in smaller variances in the treated arm. Panel E shows a different effect in 2 subgroups, but the variability is now reduced indicating that the best solution would be to identify the subpopulations in order to refine the selection criteria. In Panel F, the treatment has a stabilizing effect, with higher blood pressure falling more in severe patients, thus resulting in lower variability in the treatment arm. In the study from Kim et al.15, the primary endpoint was the PTSD Checklist–Civilian version (PCL-C). This scale is based on the sum of 17 Likert symptoms, ranging from 17 (perfect health) to 85 (worst clinical situation). At the end of the trial, the respective outcome SDs were 16 and 3 for the control and treated arms, meaning that variance was reduced around 28 times. This situation can correspond to scenarios E or F and merit much more statistical considerations, which is beyond the scope of this paper.\n\nThe assumption that the average effect equals the single unit effect underlies the rationale behind the usual sample size calculation, where only a single effect is specified. As an example, the 10 clinical trials published in the Trials Journal in October 2017 (see Supplementary File 1 : Table S1) were designed under this scenario of a fixed, constant or unique effect in the sample size calculation.\n\nOur objectives were, first, to compare the variability of the main outcome between different arms in clinical trials published in medical journals and, second, to provide a first, rough estimate of the proportion of studies that could potentially benefit from precision medicine. As sensitivity analysis, we explore the changes in the experimental arm’s variability over time (from baseline to the end of the study). We also fit a random effect model to the outcome variance ratio in order to isolate studies with a variance ratio outside their expected random variability values (heterogeneity).\n\n\nMethods\n\nOur target population was parallel randomized clinical trials with quantitative outcomes. Trials needed to provide enough information to assess two homoscedasticity assumptions in the primary endpoint: between arms at trial end; and baseline to outcome over time in the treated arm. Therefore, baseline and final SDs for the main outcome were necessary or, failing that, at least one measure that would allow us to calculate them (variances, standard errors or mean confidence intervals).\n\nArticles on parallel clinical trials from the years 2004, 2007, 2010 and 2013 were selected from the Medline database with the following criteria: “AB (clinical trial* AND random) AND AB (change OR evolution OR (difference AND baseline)” [The word “difference” was paired with “baseline” because the initial purpose of the data collection, subsequently modified, was to estimate the correlation between baseline and final measurements]. The rationale behind the election of these years was to have a global view of the behavior of the studies over a whole decade. For the years 2004 and 2007, we selected all papers that met the inclusion criteria; while for the years 2010 and 2013, as we obtained a greater number of articles retrieved from the search (478 and 653, respectively), we chose a random sample of 300 papers (Section II in Supplementary File 1).\n\nData were collected by two different researchers (NM, MkV) in two phases: 2004/2007 and 2010/2013. Later, two statisticians (JC, MtV) verified the correctness of the data and to make them accessible to readers through a shiny application and through the Figshare repository16.\n\nCollected variables were: baseline and outcome SDs; experimental and reference interventions; sample size in each group; medical field according to Web of Science (WOS) classification; main outcome; patient’s disease; kind of disease (chronic or acute); outcome type (measured or scored); intervention type (pharmacological or not); and whether or not the main effect was significant.\n\nFor studies with more than one quantitative outcome, primary endpoint was determined according to the following hierarchical criteria: (1) objective or hypothesis; (2) sample size determination; (3) main statistical method; or (4) first quantitative variable reported in results.\n\nIn the same way, the choice of the \"experimental\" arm was determined depending on the role in the following sections of the article: (1) objective or hypothesis; (2) sample size determination; (3) rationale in the introduction or (4) first comparison reported in results (in the case of more than two arms)\n\nWe assessed homoscedasticity between treatments and over time. Our main analysis compared, for the former, the outcome variability between Treated (T) and Control (C) arms at the trial end. For the latter, we compared the variability between Outcome (O) and its Baseline (B) value for the treated arm.\n\nTo distinguish between random variability and heterogeneity, we fitted a random mixed effects model using the logarithm of the variance ratio at the end of the trial as response with the study as random effect and the logarithm of the variance ratio at baseline as fixed effect17. An analogous model was employed to assess the homoscedasticity over time, as such a model allows the separation of random allocation variability from additional heterogeneity. To obtain a reference in the absence of treatment effect, we first modeled the baseline variance ratio as a response that is expected to have heterogeneity equal to 0 due to randomization – so long as no methodological impurities are present.(e.g., consider the outcomes obtained 1 month after the start of treatment as the baseline values). This reference model allows us to know the proportion of studies in the previous models that could have additional heterogeneity which cannot be explained by the variability among studies (sections III, V and VI in Supplementary File 1).\n\nFunnel plots, centered at zero, of the measurement of interest as a function of its standard errors are reported in order to investigate asymmetries.\n\nAs sensitivity analyses, we assessed homoscedasticity in each single study: (a) between outcomes on both arms with F-test for independent samples; and (b) between baseline and outcome in the treated arm with a specific test for paired samples18 when the variance of the paired difference was available. All tests were two-sided (α=5%).\n\nSeveral subgroup analyses were carried out according to the statistical significance of the main effect of the study and to the different types of outcomes and interventions.\n\nAll analyses were performed with the R statistical package version 3.2.5. (The R code for the main analysis is available from https://doi.org/10.5281/zenodo.113360919)\n\n\nResults\n\nA total of 1214 articles were retrieved from the search. Of those papers, 542 belong to the target population and 208 (38.4%) contained enough information to conduct the analysis (Figure 2).\n\nPercentages represent the quantity of papers in the target population. The number of articles for each year (2004/2007/2010/2013) is specified in the second line of each box (separated by slashes). $300 papers were randomly selected for years 2010 and 2013. Four papers were excluded because the variance of the change over time was inconsistent with both the baseline and final variances, which would lead to impossible absolute correlation estimates greater than 1.\n\nMainly, the selected studies were non-pharmacological (122, 58.6%), referred to chronic conditions (101, 57.4%), had an outcome measure with units (132, 63.8%) instead of a constructed scale, and this outcome were measured rather than assessed (125, 60.1%). Regarding the primary objective of each trial, the authors found statistically significant differences between the arms in 83 (39.9%) studies. Following the WOS criteria, 203 articles (97.6%) belonged to at least one medical field. The main areas of study were: General & Internal Medicine (n=31, 14.9%), Nutrition & Dietetics (21, 10.1%), Endocrinology & Metabolism (19, 9.1%), and Cardiovascular System & Cardiology (16, 7.7%).\n\nThere is a high average concordance between variances in the treatment and control arm, but with evidence of a smaller variability in the treated arm. At the end of the study, 113/208 (54%, 95% CI, 47 to 61%) papers showed less variability in the treated arm (Supplementary File 1 : Figure S1). Among the treated arms, 111/208 (53%, 95% CI, 46 to 60%) had less or equal variability at the end of follow-up than at the beginning (Supplementary File 1 : Figure S2).\n\nWe found statistically significant differences (at 5%) between outcome variances in 41 out of 208 (19.7%) studies: 7.2% were in favor of greater variance in the treated arm, and 12.5% in the opposite direction. A greater proportion was obtained from the comparisons over time of 95 treated arms: 16.8% had significantly greater variability at the end of the study and 23.2% at the beginning (Table 1).\n\nAlternative possible methods to estimate the number and percentage of studies with different variances on comparisons between arms and over-time. Limits for declaring different variances come from different statistical methods; either masked specified statistical tests (F for independent outcomes or Sachs’ test18 for related samples); or sensitivity analysis about the number of studies that have to be deleted from the random mixed model in order to achieve a negligible heterogeneity (see Methods for details). ¥ Only performed in studies reporting enough information to obtain the variability of the change from baseline to outcome, for example because they provide the correlation.\n\nRegarding the comparison between arms, the adjusted point estimate of the ratio (Treated to Control group) of the outcome variances is 0.89 (95% CI 0.81 to 0.97), indicating that treatments tend to reduce the variability of the patient's response by about 11% on average. The comparison over time provides a similar result: the average variability at the end of the study is 14% lower than that at the beginning (Supplementary File 1 : Table S4).\n\nAccording to the random model, the baseline heterogeneity was 0.31; this is a very high value which can only be explained by methodological flaws similar to those presented by Carlisle20. Fortunately, the exclusion of the four most extreme papers reduced it to 0.07; one of them was the study by Hsieh et al.21 whose “baseline” values were obtained 1 month after the treatment started. When we modeled the outcome instead of the baseline variances as the response, heterogeneity was approximately doubled. We found 30 studies that compromised homoscedasticity (11 with higher variance in the treated arm and 19 with lower, Table 1). Figure 3 shows the funnel plots for both between-arm and over-time comparisons.\n\nFunnel plots of variance ratio between arms with the 208 studies (Panel A) and for over-time comparison with the 95 studies for which the variance of the difference between the basal and final response was available (Panel B). Vertical axis indicates precision for the comparison of variances; with points outside the triangle being statistically significant. Additionally, red points mark significant differences between the means, which correspond to the objective of each study to assess main treatment effects. In Panel A, points on the right indicate higher outcome variability for the treated individuals, as expected if there is patient-by-treatment interaction; similarly, points on the left correspond to lower variability, although this is compatible with traditional Evidence-Based Medicine. Eleven (5.2%) out of 208 studies reported exactly the same outcome variability in both arms. We observe more red points on the left, indicating that changes in the average, come with reductions in the variance. In Panel B, points on the right indicate higher variability in the experimental arm at the end of the study, as expected in a scenario of heterogeneous treatment effect; points on the left correspond to lower variability at the end, which implies a more homogenous response after treatment. The largest number of points on the left side indicates a majority of experimental interventions that reduce variability. In addition, several of these interventions yielded significant results in the main endpoint.\n\nSubgroup analyses suggest that only significant interventions had an effect on reducing variability (Supplementary File 1 : Figures S3–S5), which has already been observed in other studies22,23 and in the line of other works that had found a positive correlation between the effect size and its heterogeneity24,25: in fact, it is difficult to find heterogeneity when there is no overall treatment effect. The remaining subgroups analyses did not raise concerns (section V in Supplementary File 1).\n\n\nDiscussion\n\nOur main objective was to show that comparing variances can provide some evidence about how much precision medicine is needed. The variability seems to decrease for treatments that perform significantly better than the reference; otherwise, it remains similar. Therefore, contrary to popular belief, variability tends to be reduced on average after treatment, thus making precision medicine dispensable in most cases. This could be due to several reasons: some measurements may have “ceiling” or “floor” effects (e.g., in the extreme case, if a treatment makes a person well, no further improvement is possible); or the treatment may act proportionally rather than linearly, in which case the logarithm of the outcome would serve as a better scale.\n\nWhen both arms have equal variances, then an obvious default explanation is that the treatment is equally effective for all, thus rendering the search for predictors of differential response futile. This means that treatment effects obtained by comparing the means between groups can be used to estimate both the averaged treatment effect and the non-observable patient treatment effect.\n\nFurthermore, our second objective was to provide a rough estimate of the proportion of interventions that require a greater degree of precision medicine, and our answer is “not many”: considering the most extreme result from Table 1, we found that 1/6 interventions (16.8%) showed some variance inflation.\n\nThere are three reasons why these findings do not invalidate precision medicine in all settings. First, some additional heterogeneity is present in the outcome variances ratio, which indicates that the variability had increased between arms as well as over time. Second, the outcomes of some type of interventions such as surgeries, for example, are greatly influenced by the skills and training of those administering the intervention, and these situations could have some effect on increasing variability. And third, we focus on quantitative outcomes, which are neither time-to-event nor binary, meaning that the effect could take a different form, such as all-or-nothing.\n\nThe results rely on published articles, which raises some relevant issues. First, some of our analyses are based on Normality assumptions for the outcomes that are unverifiable without access to raw data. Second, a high number of manuscripts (61.6%, Figure 2) act contrary to CONSORT26 advice in that they do not report variability. Thus, the results may be biased in either direction. Third, trials are usually powered to test constant effects and thus the presence of greater variability would lead to underpowered trials, non-significant results and unpublished papers. Fourth, the random effect model reveals additional heterogeneity in the outcome variances ratio, which may be the result of methodological inaccuracies20 arising from typographical errors in data translation, inadequate follow-up, insufficient reporting, or even data fabrication. On the other hand, this heterogeneity could also be the result of relevant undetected factors interacting with the treatment, which would indeed justify the need for precision medicine. A fifth limitation is that many clinical trials are not completely randomized. For example, multicenter trials are often blocked by center through the permuted blocks method. This means that if variances are calculated as if the trial were completely randomized (which is standard practice), the standard simple theory covering the random variation of variances from arm to arm is at best approximately true22\n\nThe main limitation of our study arises from the fact that, although constant effect always implies homoscedasticity on the chosen scale, the reverse is not true; i.e., homoscedasticity does not necessarily imply a constant effect. Nevertheless, a constant effect is the simplest explanation for homoscedasticity. For example, the non-parsimonious situation reflected in Figure 4 indicates homoscedasticity but without a constant effect.\n\nSBP potential values of each patient in both groups (C: Control; T: Treated) under a highly hypothetical scenario: the treatment effect has no value if systematically applied to the whole population; but if n-of-1 trials could be performed in this situation, the best treatment strategy would be chosen for each patient and the overall health of the population would be improved.\n\nHeteroscedasticity may suggest the need for further refinements of the eligibility criteria or for finding an additive scale22,27. Because interaction analyses cannot include unknown variables, all trials would potentially need to be repeated once any new potential interaction variable emerges (e.g., a new biomarker) as a candidate for a new subgroup analysis. Nevertheless, we have shown how homoscedasticity can be assessed when reporting trials with numerical outcomes, regardless of whether every potential effect modifier is known.\n\nFor most trials, subjects vary little in their response to treatment, which suggests that precision medicine’s scope may be less than what is commonly assumed. In the past century, Evidence-Based Medicine operated under the paradigm of a constant effect assumption, by which we learned from previous patients in order to develop practical clinical guides for treating future ones. Here, we have provided empirical insights for the rationale behind Evidence-Based Medicine. However, even where one common effect applies to all patients fulfilling the eligibility criteria, this does not imply the same decision is optimal for all patients, specifically because different patients and stakeholders may vary in their weighting not only of efficacy outcomes, but also of the harm and cost of the interventions – thus bridging the gap between common evidence and personalized decisions.\n\nNevertheless, in 16 trials of our sample, there was some evidence of variation arising from the treatment effect, suggesting a possible role for more tailored treatments: either with finer selection criteria (common effect within specific subgroups), or with n-of-1 trials (no subgroups of patients with a common effect). By identifying indications where the scope for precision medicine is limited, studies such as ours may free up resources for cases with a greater scope.\n\nOur results uphold the assertion by Horwitz et al. that there is a “need to measure a greater range of features to determine [...] the response to treatment”28. One of these features is an old friend of statisticians: the variance. Looking only at averages can cause us to miss out on important information.\n\n\nData availability\n\nData is available through two sources:\n\n\n\n- A shiny app that allows the user to interact with the data without the need to download it: http://shiny-eio.upc.edu/pubs/F1000_precision_medicine/\n\nThe Figshare repository: https://doi.org/10.6084/m9.figshare.555265616\n\nIn both sources, the data can be downloaded under a Creative Commons License v. 4.0.\n\nThe code for the main analysis is available in the following link: https://doi.org/10.5281/zenodo.113360919", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nPartially supported by Methods in Research on Research (MiRoR, Marie Skłodowska-Curie No. 676207); MTM2015-64465-C2-1-R (MINECO/FEDER); and 2014 SGR 464.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: The supplementary material contains the following sections:\n\nClick here to access the data.\n\n- Section I: Constant effect assumption in sample size rationale\n\n- Section II: Bibliographic review\n\n- Section III: Descriptive measures\n\n- Section IV: Random effects models\n\n- Section V: Subgroup analyses\n\n- Section VI: Standard error of log(VOT/VOC) in independent samples\n\n- Section VII: Standard error of log(VOT/VBT) in paired samples\n\n\nReferences\n\nCollins FS, Varmus H: A new initiative on precision medicine. N Engl J Med. 2015; 372: 793–-5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKohane IS: HEALTH CARE POLICY. Ten things we have to do to achieve precision medicine. Science. 2015; 349(6243): 37–8. PubMed Abstract \| Publisher Full Text\n\nSchork NJ: Personalized medicine: Time for one-person trials. Nature. 2015; 520(7549): 609–11. PubMed Abstract \| Publisher Full Text\n\nWillis JC, Lord GM: Immune biomarkers: the promises and pitfalls of personalized medicine. Nat Rev Immunol. 2015; 15(5): 323–29. PubMed Abstract \| Publisher Full Text\n\nWallach JD, Sullivan PG, Trepanowski JF, et al.: Evaluation of Evidence of Statistical Support and Corroboration of Subgroup Claims in Randomized Clinical Trials. JAMA Intern Med. 2017; 177(4): 554–60. PubMed Abstract\n\nDurán-Cantolla J, Aizpuru F, Montserrat JM, et al.: Continuous positive airway pressure as treatment for systemic hypertension in people with obstructive sleep apnoea: randomised controlled trial. BMJ. 2010; 341: c5991. PubMed Abstract \| Publisher Full Text\n\nKojima Y, Kaga H, Hayashi S, et al.: Comparison between sitagliptin and nateglinide on postprandial lipid levels: the STANDARD study. World J Diabetes. 2013; 4(1): 8–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational conference on harmonisation: statistical principles for clinical trials ICH-E9.1998. Accessed September 14 2017. Reference Source\n\nShamseer L, Sampson M, Bukutu C, et al.: CONSORT extension for reporting N-of-1 trials (CENT) 2015: Explanation and elaboration. BMJ. 2015; 350: h1793. PubMed Abstract \| Publisher Full Text\n\nAraujo A, Julious S, Senn S: Understanding Variation in Sets of N-of-1 Trials. PLoS One. 2016; 11(12): e0167167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenn S: Individual response to treatment: is it a valid assumption? BMJ. 2004; 329(7472): 966–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenn S: Mastering variation: variance components and personalised medicine. Stat Med. 2016; 35(7): 966–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang R, Lagakos SW, Ware JH, et al.: Statistics in medicine--reporting of subgroup analyses in clinical trials. N Engl J Med. 2007; 357(21): 2189–94. PubMed Abstract \| Publisher Full Text\n\nSenn S, Richardson W: The first t-test. Stat Med. 1994; 13(8): 785–803. PubMed Abstract \| Publisher Full Text\n\nKim SH, Schneider SM, Bevans M, et al.: PTSD symptom reduction with mindfulness - based stretching and deep breathing exercise: randomized controlled clinical trial of efficacy. J Clin Endocr Metab. 2013; 98(7): 2984–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCortés J: ‘review_homoscedasticity_clinical_trials’. [Data set]. 2017. Publisher Full Text\n\nBartlett MS, Kendall DG: The statistical analysis of variance-heterogeneity and the logarithmic transformation. J R Stat Soc. 1946; 8(1): 128–38. Publisher Full Text\n\nSachs L: Applied Statistics: A Handbook of Techniques. 2nd ed. New York: Springer-Verlag, 1984. Publisher Full Text\n\nCortés J: R code for analysis of homoscedasticity in clinical trials. Zenodo. 2017. Publisher Full Text\n\nCarlisle JB: Data fabrication and other reasons for non-random sampling in 5087 randomised, controlled trials in anaesthetic and general medical journals. Anaesthesia. 2017; 72(8): 944–952. PubMed Abstract \| Publisher Full Text\n\nHsieh LL, Kuo CH, Yen MF, et al.: A randomized controlled clinical trial for low back pain treated by acupressure and physical therapy. Prev Med. 2004; 39(1): 168–76. PubMed Abstract \| Publisher Full Text\n\nSenn S: controversies concerning randomization and additivity in clinical trials. Stat Med. 2004; 23(24): 3729–53. PubMed Abstract \| Publisher Full Text\n\nJamieson J: Measurement of change and the law of initial values: A computer simulation study. Educ Psychol Meas. 1995; 55(1): 38–46. Publisher Full Text\n\nSenn S: Trying to be precise about vagueness. Stat Med. 2007; 26(7): 1417–30. PubMed Abstract \| Publisher Full Text\n\nGreenlaw N: Constructing appropriate models for meta-analyses. University of Glasgow, 2010. Accessed September 14, 2017. Reference Source\n\nSchulz KF, Altman DG, Moher D, et al.: CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010; 340: c332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRothman KJ, Greenland S, Walker AM: Concepts of interaction. Am J Epidemiol. 1980; 112(4): 467–70. PubMed Abstract \| Publisher Full Text\n\nHorwitz RI, Cullen MR, Abell J, et al.: Medicine. (De)personalized medicine. Science. 2013; 339(6124): 1155–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "30604", "date": "02 Mar 2018", "name": "Ian R. White", "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 paper considers randomised trials (RCTs) of treatment versus control with a quantitative outcome. It observes that if treatment effects are homogeneous (the same for all trial participants) then the outcome variance will be the same in both trial arms. It therefore reviews the extent to which the outcome variance is the same across trial arms in 208 published RCTs. It finds 41 RCTs with significant differences in outcome variance, and that it is more common for the outcome variance to be smaller in the treatment arm rather than larger.\nMy overall comment is that the analysis results are useful, but they need to be made clearer, and the interpretation should be much more cautious. Points marked * must be addressed to make the article scientifically sound (with ** the top priority).\nBackground\nAbstract, background: “The conventional design of randomized trials assumes that each individual benefits by the same amount.” This is also asserted elsewhere in the paper, but it is not true. From a causal inference perspective, a RCT estimates the average causal effect, which is well defined in the presence of treatment effect heterogeneity. This is why the trials community worries so much about external generalisability: for example, if a trial treated 60% women and 40% men and showed a benefit of treatment, then a clinician treating women and men in the same ratio can be confident of giving a benefit overall, but a clinician treating women and men in a different ratio cannot be so confident. This point (repeated elsewhere) is not essential to the paper’s argument, so should be removed. Similarly, the argument “The assumption that the average effect equals the single unit effect underlies the rationale behind the usual sample size calculation, where only a single effect is specified” (Introduction) is false. Sample size calculations relate only to comparisons of group averages.\n\nMethods The methods used appear entirely appropriate. However they are not well described.\nTerminology must be improved. For example, the key outcome in this study is the ratio of variances between treatment and control arms, and this (or its opposite) is variously called “homoscedasticity”, “heterogeneity”, even “concordance”. The authors should choose a term and stick with it. Similarly for the “random mixed effects model” which later becomes the “random model”. (I’m going to use “homoscedasticity” and “random-effects model”.) The authors are doing a meta-analysis, even though they don’t call it that, so the term “heterogeneity” should be reserved for “variation between studies”, i.e. tau^2 in the random effects models. It’s not clear to me what the “random model” results in Table 1 are. Since this is a model across studies, how can it count individual studies? If empirical Bayes estimates of study-specific effects are being tested, this must be explained. Trials that are “significant” are combined - “Subgroup analyses suggest that only significant interventions had an effect on reducing variability” - but interventions that increase the mean should be separated from those that decrease the mean. The later conclusion that “The variability seems to decrease for treatments that perform significantly better than the reference” suggests a different distinction (better/worse is not the same as larger/smaller because outcomes may be positive or negative) and is not supported by the results presented. Abstract, Results: “The adjusted point estimate of the mean ratio (treated to control group) of the outcome variances” is not clear without reading the whole text. Again, defining a term (“outcome variance ratio”?) will help. Table 1, “variability is… increased”: from the text, this means “significantly increased”, which should be clarified.\n\nInterpretation The results may be interpreted in many ways, which are sensibly discussed by the authors. Most importantly, treatment effect homogeneity implies homoscedasticity, but the converse (“homoscedasticity implies treatment effect homogeneity”) is not true: this is demonstrated very nicely in Figure 4. Homoscedasticity is scale-dependent: for example, it may be removed (or created) by a log transformation (mentioned in the Discussion).\nThe authors omit one alternative explanation of homoscedasticity over time: clinical trial populations have eligibility criteria at baseline which may limit baseline variance. For example, a hypertension trial might recruit patients with baseline SBP between 140 and 159 mm Hg. In this case, variance is very likely to naturally increase over time. *The authors’ conclusions ignore the alternative interpretations noted above. Here are some examples which are illogical:\nAbstract, Conclusions: “the variance was more often smaller in the intervention group, suggesting, if anything, a reduced role for precision medicine”, and Discussion: “variability tends to be reduced on average after treatment, thus making precision medicine dispensable in most cases”. This is actually false. If a study finds smaller variance in the treated group then we DO have evidence of treatment effect heterogeneity, and indeed the treatment may be doing exactly what medicine should do - making the sickest better while not harming the less sick. Introduction: “If this homoscedasticity holds, there is no need to repeat the clinical trial once a new possible effect modifier becomes measurable” - again, this wrongly assumes the converse stated above. Discussion: “When both arms have equal variances, then an obvious default explanation is that the treatment is equally effective for all, thus rendering the search for predictors of differential response futile”: this is illogical. Discussion: “For most trials, subjects vary little in their response to treatment, which suggests that precision medicine’s scope may be less than what is commonly assumed.” : this is also illogical.\n\nIn the light of the above arguments, I find the statement (Abstract, Conclusions) that “Homoscedasticity is a useful tool for assessing whether or not the premise of constant effect is reasonable” to be highly debatable. Logic suggests it gives a lower bound on the extent of usefulness of precision medicine, and the results of this study do not add any more to this. The objectives in the Discussion should be the same as those stated in the Introduction.\n\nSource data\nI had trouble opening the source data both in Excel (since the csv file is in fact semi-colon-delimited) and in Stata (which was thrown by line 80). Could it be provided in a more convenient format or with some notes?\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? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3701", "date": "13 Jun 2018", "name": "Jordi Cortés", "role": "Author Response", "response": "JOINT ANSWER to Ian White and Saskia le CessieThis is a general response to Ian White and Saskia Le Cessie on why we stated that the standard clinical trial design and analysis assume a constant effect.In the following, (1) we update the standard sample size rationale; and (2) we explain why inflated variances may require precision medicine in just two general cases: (a) interaction, as represented in Fig. 1, panel C; and (b) random treatment effect, Fig. 1, panel D. Under the Neyman-Pearson framework to determine sample size, a single effect size value Δ is specified under the alternative hypothesis H1, assuming in that way a constant effect, as in Fig. 1, panels A (H0) and B (H1). We devise two situations that, because they result in higher variance, they would need personalized medicine: Interaction between treatment and a baseline variable such us, for example, gender (Fig. 1, panel C). In this scenario there are two subpopulations (e.g., men and women) with different treatment effects that require the effect to be made further “precise”. Random treatment effect on each patient (Fig. 1, panel D). In this scenario, the effect size does not depend on a known patient baseline characteristic and the only way to estimate the individual patient effect is by means of individualized trials (“n of 1” trials). Those 2 hypothetical scenarios, lead to an increased variance. Conversely, scenarios E and F represent two similar situations (interaction and random effect) but result in reduced variance –without relevant changes on the average. Although we agree that in those two last scenarios leading to reduced variability the specific patient treatment effect may still be unknown because the outcome has reduced variability with a similar central overall position, we argue that patients in those situations were subject to “further control” (having more stable values within the boundaries of “normality”). So, the usual sample size rationale specified by statisticians in trials assumes a constant, unique effect that agrees with the clinical and legal interpretation that the effect is the same – or at least similar enough to be considered homogeneous – for all the patients fulfilling the eligibility criteria. To illustrate this secondary “argument”, we reviewed the sample size rationale for the last (at that time) 10 protocols published in Trials, and we found that all of them defined a single effect size (100%, two-sided 95% confidence interval from 69% to 100%). In addition, we have included a new column in Table S1 with the main analysis showing that the SAP in all those cases (10 out of 10, 95%CI from 69 to 100%) was also designed to estimate a single, constant effect.We have modified Fig. 1 (panels E and F) to show decreasing variance treatment effects, but now without affecting the average. We have also improved the 2 following sentences:Before [Abstract]: However, the conventional design of randomized trials assumes that each individual benefits by the same amount.After [Abstract]: However, conventional clinical trials are designed to find differences with the implicit assumption that the effect is the same in all patients within the eligibility criteria.Before [Introduction]: The assumption that the average effect equals the single unit effect underlies the rationale behind the usual sample size calculation, where only a single effect is specified. As an example, the 10 clinical trials published in the Trials Journal in October 2017 (see Supplementary material: Table S1) were designed under this scenario of a fixed, constant or unique effect in the sample size calculation.After [Introduction]: The assumption of homoscedasticity in the usual calculations of sample size is better interpreted under the constant effect model (Figure 1, panels A, H0; and B, H1). As an example, the 10 clinical trials published in the Trials Journal in October 2017 (Table S1 of Supplementary material) were designed with only a constant for the effect size. Furthermore, all their analyses were designed to test (and estimate) a single constant for the effect size. In other words, there was mention of neither any possible interaction with baseline variables (Figure 1, scenarios C and E), nor of any random variability for the treatment effect (Figure 1, scenarios D and F); and thus, all those trials were designed to test a constant effect.We have also updated the legend of Figure 1 to highlight that now panels C to F show only possible individual treatment effects on variances but not on means.We are deeply grateful to Ian White and Saskia le Cessie for highlighting the need to clarify this crucial issue. Ian WhiteFrom here, we’ll answer specific issues This paper considers randomised trials (RCTs) of treatment versus control with a quantitative outcome. It observes that if treatment effects are homogeneous (the same for all trial participants) then the outcome variance will be the same in both trial arms. It therefore reviews the extent to which the outcome variance is the same across trial arms in 208 published RCTs. It finds 41 RCTs with significant differences in outcome variance, and that it is more common for the outcome variance to be smaller in the treatment arm rather than larger.My overall comment is that the analysis results are useful, but they need to be made clearer, and the interpretation should be much more cautious. Points marked must be addressed to make the article scientifically sound (with ** the top priority). We are grateful to Prof. Ian White for his suggestions, which will definitively help us to improve our manuscript. Background1.Abstract, background: “The conventional design of randomized trials assumes that each individual benefits by the same amount.” This is also asserted elsewhere in the paper, but it is not true. From a causal inference perspective, a RCT estimates the average causal effect, which is well defined in the presence of treatment effect heterogeneity. This is why the trials community worries so much about external generalisability: for example, if a trial treated 60% women and 40% men and showed a benefit of treatment, then a clinician treating women and men in the same ratio can be confident of giving a benefit overall, but a clinician treating women and men in a different ratio cannot be so confident. This point (repeated elsewhere) is not essential to the paper’s argument, so should be removed.2. Similarly, the argument “The assumption that the average effect equals the single unit effect underlies the rationale behind the usual sample size calculation, where only a single effect is specified” (Introduction) is false. Sample size calculations relate only to comparisons of group averages.Thanks again for highlighting this hugely important issue. We have addressed these two comments in the previous common answer. MethodsThe methods used appear entirely appropriate. However they are not well described.1. Terminology must be improved. For example, the key outcome in this study is the ratio of variances between treatment and control arms, and this (or its opposite) is variously called “homoscedasticity”, “heterogeneity”, even “concordance”. The authors should choose a term and stick with it. Similarly for the “random mixed effects model” which later becomes the “random model”. (I’m going to use “homoscedasticity” and “random-effects model”.)Thanks. To simplify the notation, we have deleted the term “concordance”. We also reserved the term heterogeneity for the tau^2 statistic resulting from the mixed-effects model (see next answer). Furthermore, we have homogenized the terms for referring to the “random-effects model” throughout the text. 2. The authors are doing a meta-analysis, even though they don’t call it that, so the term “heterogeneity” should be reserved for “variation between studies”, i.e. tau^2 in the random effects models.We appreciate this insightful observation. In the random-effects model, we measured heteroscedasticity with the mu parameter, and heterogeneity between studies, through tau^2. In order to clarify this as much as possible, we have specified in the Methods section that tau^2 is used for measuring heterogeneity; and this has also been included between brackets in the Results section:“The estimated value of tau^2 provides a measure of heterogeneity, that is, to what extent the value of mu is applicable to all studies. The larger tau^2 is, the less the homogeneity”3. It’s not clear to me what the “random model” results in Table 1 are. Since this is a model across studies, how can it count individual studies? If empirical Bayes estimates of study-specific effects are being tested, this must be explained.We used the Delta method to estimate the within study variability (specifically, the variance of the logarithm of the outcome variance ratio). We have included this explanation in the Methods section: “As there is only one available measure for each study, both sources of variability cannot be empirically differentiated: (i) within study or random or that one related to sample size; and (ii) heterogeneity. In order to isolate the second, the first was theoretically estimated using the Delta method –as explained in Sections V and VI of Supplementary material“4. Trials that are “significant” are combined - “Subgroup analyses suggest that only significant interventions had an effect on reducing variability” - but interventions that increase the mean should be separated from those that decrease the mean. The later conclusion that “The variability seems to decrease for treatments that perform significantly better than the reference” suggests a different distinction (better/worse is not the same as larger/smaller because outcomes may be positive or negative) and is not supported by the results presented.Thanks for this great contribution. Following your suggestion, we have sought in each primary endpoint for whether improvements in the response correspond to higher (e.g., mobility) or lower (e.g., pain) values. This new factor has been included in the subgroup analysis (see new figures S5-S7 clicking here or in the Supplementary Material), thus providing an argument for the existence of a \"floor\" effect in those studies where a lower value corresponds to a better condition. We have added an interpretation of this finding in the Discussion:“This reduced variability could also be due to methodological reasons. One is that some measurements may have a “ceiling” or “floor” effect (e.g., in the extreme case, if a treatment heals someone, no further improvement is possible). In fact, according to the subgroup analysis of the studies with outcomes that indicate the degree of disease (high values imply greater severity; e.g., pain), a greater variance (25%) is obtained in the experimental arm (see Figure S5). However, in the studies with outcomes that measure the degree of healthiness (high values imply better condition; e.g., mobility), the average variances match between arms and do not suggest a ceiling effect.”In addition, we have included this new factor (direction of the improvement) in the Shiny app.On the other hand, all the significant studies were in favor of the experimental group; therefore, in our context, \"statistically significant\" is equivalent to \"better response in the experimental group\". We have specified this statement in the manuscript and we have kept the sentence: \"the authors found statistically significant differences between the arms (all of them in favor of the experimental group) in 83 (39.9%) studies\"5. Abstract, Results: “The adjusted point estimate of the mean ratio (treated to control group) of the outcome variances” is not clear without reading the whole text. Again, defining a term (“outcome variance ratio”?) will help.Thanks. Corrected both in the Abstract and the main text:Before [Abstract]: We assessed homoscedasticity by comparing the outcome variability between treated and control armsAfter [Abstract]: We assessed homoscedasticity by comparing the variance of the primary endpoint between arms through the outcome variance ratio (treated to control group).Before [Abstract]: The adjusted point estimate of the mean ratio (treated to control group)After [Abstract]: The adjusted point estimate of the mean outcome variance ratio (treated to control group) …Before [Methods]: … we fitted a random-mixed effects model using the logarithm of the variance ratio at the end of the trial…After [Methods]: … we fitted a random-effects model using the logarithm of the outcome variance ratio at the end of the trial …6. Table 1, “variability is… increased”: from the text, this means “significantly increased”, which should be clarified.Thanks. We have corrected it:Before [Table 1]: increased/decreasedAfter [Table 1]: significantly increased / significantly decreasedInterpretationThe results may be interpreted in many ways, which are sensibly discussed by the authors.Most importantly, treatment effect homogeneity implies homoscedasticity, but the converse (“homoscedasticity implies treatment effect homogeneity”) is not true: this is demonstrated very nicely in Figure 4. Homoscedasticity is scale-dependent: for example, it may be removed (or created) by a log transformation (mentioned in the Discussion).1. The authors omit one alternative explanation of homoscedasticity over time: clinical trial populations have eligibility criteria at baseline which may limit baseline variance. For example, a hypertension trial might recruit patients with baseline SBP between 140 and 159 mm Hg. In this case, variance is very likely to naturally increase over time.Thanks again. We have dealt with this in the Discussion:“…it has been observed that the variability in the experimental arm also decreases from baseline to the end of the study, although this comparison is not protected by randomization; for example, the existence of eligibility criteria at baseline may have limited the initial variance (a hypertension trial might recruit patients with baseline SBP between 140 and 159 mm Hg), leading to the variance naturally increasing over time”2. *The authors’ conclusions ignore the alternative interpretations noted above. Here are some examples which are illogical: Abstract, Conclusions: “the variance was more often smaller in the intervention group, suggesting, if anything, a reduced role for precision medicine”, and Discussion: “variability tends to be reduced on average after treatment, thus making precision medicine dispensable in most cases”. This is actually false. If a study finds smaller variance in the treated group then we DO have evidence of treatment effect heterogeneity, and indeed the treatment may be doing exactly what medicine should do - making the sickest better while not harming the less sick. Thanks. We agree. We have addressed this point in the general response above. We provide here further specific comments.There is heteroscedasticity of effect leading to reduced outcome variability, such as the one shown in examples E and F of Figure 1. Those cases with reduced variability show situations in which the outcome is “under additional control” at the end. The only mathematical model that we can imagine here is the one with an effect correlated with baseline values: higher effects for higher (worse) baseline values. We can imagine this situation for the “ideal” training program: worse participants at the beginning, which further increases or reduces variability. So, although we agree that this is a theoretical heterogeneity, we do not think that it has any practical implication for “individualizing” the treatment: all patients benefit (although to a different degree) from the intervention; and at the end, all patients are “under additional control”.We have performed some changes in the manuscript in order to clarify this point:Before [Abstract]: the variance was more often smaller in the intervention group, suggesting, if anything, a reduced role for precision medicineAfter [Abstract]: We found that the outcome variance was more often smaller in the intervention group, suggesting that treated patients may end up pertaining more often to reference or “normality” values and thus would not require further precision medicine. However, this result may also be compatible with a reduced effect in some patients, which would require studying whether the effect merits enduring the side effects as well as the economic costs. Before [Discussion]: variability tends to be reduced on average after treatment, thus making precision medicine dispensable in most casesAfter [Discussion]: We found that variability seems to decrease for treatments that perform significantly better than the reference; otherwise, it remains similar. Therefore, the treatment seems to be doing what medicine should do –having larger effects in the most ill patients. Two considerations may be highlighted here: (1) as the outcome range becomes reduced, we may interpret that, following the intervention, this population is under additional control; but also, (2) as subjects are responding differently to treatment, this opens the way for not treating some (e.g. those subjects who are not very ill, and so have no scope to respond very much), with obvious savings in side effects and costs Introduction: “If this homoscedasticity holds, there is no need to repeat the clinical trial once a new possible effect modifier becomes measurable” - again, this wrongly assumes the converse stated above. In this case, we have softened the sentence by changing the term \"need\" to \"evidence\".Before [Introduction]: If this homoscedasticity holds, there is no need to repeat the clinical trial once a new possible effect modifier becomes measurableAfter [Introduction]: If this homoscedasticity holds, there is no evidence that the clinical trial should be repeated once a new possible effect modifier becomes measurable Discussion: “When both arms have equal variances, then an obvious default explanation is that the treatment is equally effective for all, thus rendering the search for predictors of differential response futile”: this is illogical. We are not sure that we understood why this is illogical. Anyway, we have softened the sentence by changing \"an obvious default explanation\" to \"the simplest explanation\".Before [Discussion]: When both arms have equal variances, then an obvious default explanation is that the treatment is equally effective for all, thus rendering the search for predictors of differential response futileAfter [Discussion]: When both arms have equal variances, then the simplest explanation is that the treatment is equally effective for all, thus rendering the search for predictors of differential response futile. Discussion: “For most trials, subjects vary little in their response to treatment, which suggests that precision medicine’s scope may be less than what is commonly assumed”: this is also illogical. Again, we are not sure that we understood why this is illogical. Nevertheless, we have referred to the limitations derived from Figure 4.Before [Discussion]: For most trials, subjects vary little in their response to treatment, which suggests that precision medicine’s scope may be less than what is commonly assumedAfter [Discussion]: For most trials, variability of the response to treatment changes scarcely or even decreases, which suggests that precision medicine’s scope may be less than what is commonly assumed – while always taking into account the limitation previously explained in Figure 4.3. In the light of the above arguments, I find the statement (Abstract, Conclusions) that “Homoscedasticity is a useful tool for assessing whether or not the premise of constant effect is reasonable” to be highly debatable. Logic suggests it gives a lower bound on the extent of usefulness of precision medicine, and the results of this study do not add any more to this.We have reduced the ostentatious nature of this phrase, warning the reader that there are limitations to this methodology:“We have shown that the comparison of variances is a useful but not definitive tool to asses if the design assumption of a constant effect holds.”4. *The objectives in the Discussion should be the same as those stated in the Introduction.Thanks. We have simplified the objectives in the introduction:Before: Our objectives were, first, to compare the variability of the main outcome between different arms in clinical trials published in medical journals and, second, to provide a first, rough estimate of the proportion of studies that could potentially benefit from precision medicine. As sensitivity analysis, we explore the changes in the experimental arm’s variability over time (from baseline to the end of the study). We also fit a random- effects model to the outcome variance ratio in order to isolate studies with a variance ratio outside their expected random variability values (heterogeneity).After: Our objectives were, first, to compare the variability of the main outcome between different arms in clinical trials published in medical journals using a random-effects model; and, second, to provide a rough estimate of the proportion of studies that could potentially benefit from precision medicine. Finally, we explore the changes in the experimental arm’s variability over time (from baseline to the end of the study). Also, we have reordered the whole Discussion section according to these objectives: 1) Variability comparison between arms and explanation 2) Rough estimate of the studies that potentially benefit from precision medicine (greater variability in experimental arms) 3) Variability comparison between arms and explanation provided in your first suggestion of this section. Source data1. I had trouble opening the source data both in Excel (since the csv file is in fact semi-colon-delimited) and in Stata (which was thrown by line 80). Could it be provided in a more convenient format or with some notes?We have changed the format (now, columns are comma-delimited) both in the Shiny app and in the Figshare repository. We also solved the problem with line 80, which included some unnecessary quotation marks (“) in the Title field." }, { "c_id": "4182", "date": "15 Nov 2018", "name": "Jordi Cortés", "role": "Author Response", "response": "Following your suggestions together with those of the other reviewers, we have updated the manuscript with a new version that aims to emphasize the fact that researchers’ assumption of a constant effect is not clear as long as they do not mention it explicitly. Admittedly, in those studies whose sample size calculation considers some variability in the treatment effect, there is no doubt that this premise has not been considered." } ] }, { "id": "31694", "date": "23 Mar 2018", "name": "Erica E.M. Moodie", "expertise": [ "Reviewer Expertise Longitudinal data analysis", "adaptive treatment strategies" ], "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 have performed a review of a sample of clinical trials conducted every three years from 2004-2013 to examine whether there exists post-treatment heterogeneity in participants responses with premise that lack of heterogeneity suggests that precision medicine is not warranted.\n\nWhile the question is one that should be asked. However, the study carried out is not suited to answering the question as it has been conducted in randomized trials where there is typically little heterogeneity. That is, the authors have performed a perfectly reasonable analysis that cannot answer the pertinent question. It is well known that randomized trials tend to be populated by homogenous population (more white, more male, etc.) – see, for example Oh et al. (2015) Diversity in Clinical and Biomedical Research: A Promise Yet to Be Fulfilled. PLoS Med 12(12): e1001918, Caplan & Friesen P (2017) Health disparities and clinical trial recruitment: Is there a duty to tweet? PLoS Biol 15(3): e2002040 and the references therein – or indeed many other papers on this topic. This may be in part a function of recruitment strategies and also by design, as trialists (particularly those testing new therapies) often determine inclusion criteria to target the (potentially homogeneous) segment of the population who might show the greatest response to the treatment. Thus, the authors have chosen to study a population that is likely to be homogeneous and not reflective of real-world clinical care. There are numerous examples covariate-tailored treatment algorithms, from the choice of hormonal therapies for women diagnosed with estrogen-receptor-positive, HER2-negative breast cancer to the choice of ACE inhibitors vs. calcium channel blockers for hypertension, that the authors choose to overlook as cases where we have learned about previous patients with particular characteristics to learn about future similar patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3699", "date": "04 Jun 2018", "name": "Jordi Cortés", "role": "Author Response", "response": "The referee’s objections can be summarized in two points:(1) “There are numerous examples of covariate-tailored treatment algorithms.”(2) “Randomized trials tend to be populated by homogenous populations”, which in turn does not reflect a real population’s existing variability.We understand the reviewer’s comments, but we disagree with the reviewer’s conclusions:(1) Our work does not intend to completely invalidate precision medicine. We are not stating that there are not “examples of covariate-tailored treatments”; rather that (Abstract): “We found that the outcome variance was more often smaller in the intervention group, suggesting that treated patients may end up pertaining more often to reference or normality values and thus would not require further precision medicine”. This was already stated in the discussion: “these findings do not invalidate precision medicine in all settings.” Thus in the quite wide settings of our trials, we found little evidence that precision medicine would be of any use.(2) The referee argues that trials have “too many” selection criteria to reflect “a real population”. This is a standard criticism of explanatory clinical trials, suggesting that the selection criteria are usually “too many”. And we agree, because our point is that most trials have “enough” selection criteria to provide a homogeneous effect. Furthermore, we also agree that we can only talk about “published trials with eligibility criteria”. As for whether those selection criteria should be used to define the target population in clinical guidelines, there is no further need to tailor precision medicine.Dr. Moodie argues that our results do not answer the question that is posed. We also disagree. The issue of heterogeneity is obviously one that bedevils the generalizability of clinical trials. However, these are randomized comparisons; so, in the absence of a treatment effect we would expect the two arms to be comparable, no matter how heterogeneous the underlying population. The fact that even in the presence of a treatment effect there was little evidence of heterogeneity suggests there will be little scope for precision medicine in these populations. One might argue that with a more heterogeneous population, there is more scope to detect the few non-responders who would not form part of a general trial population. This does not invalidate our results; rather it argues for much larger trials with a more heterogeneous population. The point is that in the absence of these the evidence base for precision medicine is weak." } ] }, { "id": "31692", "date": "03 Apr 2018", "name": "Saskia le Cessie", "expertise": [ "Reviewer Expertise Medical Statistics", "Epidemiology", "Methods for Observational studies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview report. Does evidence support the high expectations placed in precision medicine? A bibliographic review. By J Cortés et al,\nSummary: a review of randomised trials with continuous outcomes, measured at baseline and at follow up has been conducted. The aim was to compare the variance of the outcome measure at baseline and follow-up and to compare the variances at follow-up between the treated and the control group. The authors argue that a difference in variances may indicate a heterogeneous treatment effect. General impression. The paper is well written and the results are of interest. The interpretation of the results is somewhat speculative but the authors discuss adequately the limitations. Remarks\nAbstract, Background : “However, the conventional design of randomized trials assumes that each individual benefits by the same amount.” This is not a correct statement. In a randomised trial, the average treatment effect in the population is estimated, and no assumptions about homogeneity of treatment effects are made here. The authors probably mean that many researchers implicitly assume a homogeneous treatment effect when conducting a randomised trial, interpreting the average treatment effect in the population as treatment effect at an individual level.\n\nIntroduction. I liked Figure 1 with the different explanations.\n\nMethods and flow chart. The target population was parallel randomized clinical trials with quantitative/numerical outcomes. This is not true: trials with a survival time as outcome are also trials with a numerical (sometimes censored) outcome, but are not into the scope of your paper. So please mention that you are interested in trials with a numerical response variable which are measured both at baseline and at followup. In the Flow-chart, please check whether there were indeed 150 trials with a qualitative outcome, or whether there were 150 trials which did not satisfy the requirement of both having a baseline and a followup numerical measurement.\n\nStatistical analysis. Here I got lost, the random mixed effects models should distinguish between random variability and heterogeneity, but how was unclear to me. Is adding the variance ratio at baseline needed to correct for the random variability? More details of the models and explanation of the different estimates of the model is needed, and should not only be given in the supplementary material.\n\nDid you compare the Var(change) between the treated and control group? Power to detect differences here would be larger.\n\nIt may be of interest to perform a subgroup analysis in the studies where control is placebo\n\nSupplementary material, section 4. The model has two random effects: s_i,\n\nthe heterogeneity between-study effect and e_i the within sample error with variance nu^2 . I guess this should be nu_i, as each study has its own within sample error variance, estimated form the sample sizes in the two groups (as described in the material)?\n\nThe supplementary material did not clearly described which parameter(s) from the models reflected the heterogeneity. From the main text I derived that you used the mean effect mu to indicate the amount of heterogeneity. But then how to interpret the parameter tau?\n\nSupplementary Table S4. Why not put this Table in Section 4, and make one overview of all the models fitted? And I guess that e_ij should be e_i here.\n\nResults: I did not find Figure S1 and Figure S2 very informative. Why not just give a histogram of log(var_OT/var_CT) etc.\n\nTable 1: How were the results from the random model obtained (the 11 increased, 19 decreased etc)?\n\nFigure 3. Please explain what V_OT, V_OC etc is, as Figures should be self-explained.\n\nI did not understand the second paragraph of the discussion. I guess that you want to say that the average treatment effect can be interpreted as an individual treatment effect, but I was confused at first by the words “non-observable patient treatment effect”.\n\nShocking to see that so many studies do not report measures of variability.\n\nThe fourth limitation: “the random effect model reveals additional heterogeneity”. To which result are you referring here, comparisons at baseline, followup or over time? The estimate of tau? Why should this be the result of methodological accuracies?\n\nFigure G is of interest because this is a situation where precision medicine is of interest: for some patients treatment T would be a better choice, for others treatment C and by performing precision medicine the subgroups with different responses could be detected and tailored prescriptions could be given. This indicates that observed homoscedasticity in a study should be interpreted with care and background knowledge of a study is needed to assess whether a situation as in Figure 4 is plausible.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3700", "date": "13 Jun 2018", "name": "Jordi Cortés", "role": "Author Response", "response": "JOINT ANSWER to Ian White and Saskia le CessieThis is a general response to Ian White and Saskia Le Cessie on why we stated that the standard clinical trial design and analysis assume a constant effect.In the following, (1) we update the standard sample size rationale; and (2) we explain why inflated variances may require precision medicine in just two general cases: (a) interaction, as represented in Fig. 1, panel C; and (b) random treatment effect, Fig. 1, panel D. Under the Neyman-Pearson framework to determine sample size, a single effect size value Δ is specified under the alternative hypothesis H1, assuming in that way a constant effect, as in Fig. 1, panels A (H0) and B (H1). We devise two situations that, because they result in higher variance, they would need personalized medicine: Interaction between treatment and a baseline variable such us, for example, gender (Fig. 1, panel C). In this scenario there are two subpopulations (e.g., men and women) with different treatment effects that require the effect to be made further “precise”. Random treatment effect on each patient (Fig. 1, panel D). In this scenario, the effect size does not depend on a known patient baseline characteristic and the only way to estimate the individual patient effect is by means of individualized trials (“n of 1” trials). Those 2 hypothetical scenarios, lead to an increased variance. Conversely, scenarios E and F represent two similar situations (interaction and random effect) but result in reduced variance –without relevant changes on the average. Although we agree that in those two last scenarios leading to reduced variability the specific patient treatment effect may still be unknown because the outcome has reduced variability with a similar central overall position, we argue that patients in those situations were subject to “further control” (having more stable values within the boundaries of “normality”). So, the usual sample size rationale specified by statisticians in trials assumes a constant, unique effect that agrees with the clinical and legal interpretation that the effect is the same – or at least similar enough to be considered homogeneous – for all the patients fulfilling the eligibility criteria.To illustrate this secondary “argument”, we reviewed the sample size rationale for the last (at that time) 10 protocols published in Trials, and we found that all of them defined a single effect size (100%, two-sided 95% confidence interval from 69% to 100%). In addition, we have included a new column in Table S1 with the main analysis showing that the SAP in all those cases (10 out of 10, 95%CI from 69 to 100%) was also designed to estimate a single, constant effect.We have modified Fig. 1 (panels E and F) to show decreasing variance treatment effects, but now without affecting the average. We have also improved the 2 following sentences:Before [Abstract]: However, the conventional design of randomized trials assumes that each individual benefits by the same amount.After [Abstract]: However, conventional clinical trials are designed to find differences with the implicit assumption that the effect is the same in all patients within the eligibility criteria.Before [Introduction]: The assumption that the average effect equals the single unit effect underlies the rationale behind the usual sample size calculation, where only a single effect is specified. As an example, the 10 clinical trials published in the Trials Journal in October 2017 (see Supplementary material: Table S1) were designed under this scenario of a fixed, constant or unique effect in the sample size calculation.After [Introduction]: The assumption of homoscedasticity in the usual calculations of sample size is better interpreted under the constant effect model (Figure 1, panels A, H0; and B, H1). As an example, the 10 clinical trials published in the Trials Journal in October 2017 (Table S1 of Supplementary material) were designed with only a constant for the effect size. Furthermore, all their analyses were designed to test (and estimate) a single constant for the effect size. In other words, there was mention of neither any possible interaction with baseline variables (Figure 1, scenarios C and E), nor of any random variability for the treatment effect (Figure 1, scenarios D and F); and thus, all those trials were designed to test a constant effect.We have also updated the legend of Figure 1 to highlight that now panels C to F show only possible individual treatment effects on variances but not on means.We are deeply grateful to Ian White and Saskia le Cessie for highlighting the need to clarify this crucial issue.Saskia le CessieFrom here, we’ll answer specific issues Summary: a review of randomised trials with continuous outcomes, measured at baseline and at follow up has been conducted. The aim was to compare the variance of the outcome measure at baseline and follow-up and to compare the variances at follow-up between the treated and the control group. The authors argue that a difference in variances may indicate a heterogeneous treatment effect.General impression. The paper is well written and the results are of interest. The interpretation of the results is somewhat speculative but the authors discuss adequately the limitations.Remarks We are grateful to Prof. Saskia le Cessie for her suggestions, which definitively help us to improve our manuscript. 1. Abstract, Background : “However, the conventional design of randomized trials assumes that each individual benefits by the same amount.” This is not a correct statement. In a randomised trial, the average treatment effect in the population is estimated, and no assumptions about homogeneity of treatment effects are made here. The authors probably mean that many researchers implicitly assume a homogeneous treatment effect when conducting a randomised trial, interpreting the average treatment effect in the population as treatment effect at an individual level.Yes, our impression is that at least some trialists are not aware of these assumptions. But the fact that we wanted to highlight is that trials are usually designed to provide evidence for just one parameter (in our context the “effect size” collected by the difference of means) without further specification, neither in the sample size rationale nor in the analysis of the further parameters required by precision medicine. We have addressed this point in the joint answer above.2. Introduction. I liked Figure 1 with the different explanations.Thank you. Please note that we have now updated panels C to F to isolate changes just in variance.3. Methods and flow chart. The target population was parallel randomized clinical trials with quantitative/numerical outcomes. This is not true: trials with a survival time as outcome are also trials with a numerical (sometimes censored) outcome, but are not into the scope of your paper. So please mention that you are interested in trials with a numerical response variable which are measured both at baseline and at follow-up. In the Flow-chart, please check whether there were indeed 150 trials with a qualitative outcome, or whether there were 150 trials which did not satisfy the requirement of both having a baseline and a follow-up numerical measurement.Thanks. We fully agree that discussion was introduced too late, and we have further clarified it in the Methods section and in the flow chart. The modifications are described below.Before [Methods]: Our target population was parallel randomized clinical trials with quantitative outcomesAfter [Methods]: Our target population was parallel randomized clinical trials with quantitative outcomes (not including time-to-event studies)Before [Flow chart]: Qualitative outcomeAfter [Flow chart]: Categorical or time-to-event outcome 4. Statistical analysis. Here I got lost, the random mixed effects models should distinguish between random variability and heterogeneity, but how was unclear to me. Is adding the variance ratio at baseline needed to correct for the random variability? More details of the models and explanation of the different estimates of the model is needed, and should not only be given in the supplementary material.Thanks. The model includes the (logarithm of the) baseline variances ratio because some imbalances in the initial variability between groups (after randomization) can occur simply by chance. It is foreseeable that these baseline differences in variability may influence the final differences in variability. This baseline log-ratio was highly significant (p < 0.0001) in the model. All your suggestions related to the statistical analysis (4, 7, 8, 9 and 11) and the random effects model have been addressed through a clearer and longer explanation of the model in the statistical analysis section (detailed here and in the manuscript)Nevertheless, we provide the following rule of thumb for interpreting the parameters μ (heteroscedasticity) and I2 (heterogeneity) of the random-effects model.mu < 0 --> On average, studies have lower variability in the experimental arm.mu > 0 --> On average, studies have greater variability in the experimental arm. I^2 < 25% --> As the point estimate of heterogeneity is not high enough, mu is constant throughout all the studies.I^2 < 25% --> As the point estimate of heterogeneity is high, mu does not apply to every single study. mu < 0 & I^2 < 25% --> Not one study requires precision medicine.mu < 0 & I^2 > 25% --> Some studies require precision medicine.mu > 0 & I^2 < 25% --> All studies require precision medicine.mu > 0 & I^2 > 25% --> Most studies require precision medicine.[The threshold of 25% for I^2 is based on PRISMA Statement [1] that considers values under this cutpoint as low.]The estimates of these parameters in our data were mu = -0.12 and I^2 = 80.8%, which implies that some studies require precision medicine. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche P, et al. (2009) The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: Explanation and elaboration. PLoS Med 6: e1000100. 5. Did you compare the Var(change) between the treated and control group? Power to detect differences here would be larger.Strongly agree. For high correlations between baseline and outcome, it follows that V (log (VOx/VBx)) < V (log (VOx)), as can be seen in Appendix VII of the supplementary material. However, just 95 out of 208 studies provide the Var(change) or the baseline-final correlation that would allow this analysis. 6. It may be of interest to perform a subgroup analysis in the studies where control is placeboIn fact, we performed this subgroup analysis beforehand without obtaining relevant results. We decided not to include or mention it because the distinction between a treatment called \"placebo\" and an \"active\" treatment is not clear. “Placebo” is defined as a simulator of the experimental treatment that tries to emulate its characteristics; but in some studies “control” may equal “best medical treatment”, which is also provided to “treated” patients, such that “Placebo” is complemented by the standard intervention. Because of this ambiguity in the classification, we decided to omit this information. As illustrative examples, we mention the following included studies: - Ghaleiha A, Mohammadi E, Mohammadi M, et al. Riluzole as an adjunctive therapy to risperidone for the treatment of irritability in children with autistic disorder: a doubleblind, placebocontrolled, randomized trial. Paediatr Drugs 2013; 15:505–514. The patients in the reference group took placebo in addition to risperidone (titrated up to 2 or 3 mg/day based on bodyweight) for 10 weeks.- Carroll MW, Jeon D, Mountz JM, et al. Efficacy and safety of metronidazole for pulmonary multidrug-resistant tuberculosis. Antimicrob Agents Chemother 2013; 57:3903-9. The patients in the reference group took placebo for 8 weeks in addition to an individualized background regimen.7. Supplementary material, section 4. The model has two random effects: s_i, the heterogeneity between-study effect and e_i the within sample error with variance nu^2 . I guess this should be nu_i, as each study has its own within sample error variance, estimated form the sample sizes in the two groups (as described in the material)?Thank you. We have corrected the typo including the subscript both in the Methods section and in the supplementary material: “nu_i”8. The supplementary material did not clearly describe which parameter(s) from the models reflected the heterogeneity. From the main text I derived that you used the mean effect mu to indicate the amount of heterogeneity. But then how to interpret the parameter tau?Thanks. Tau reflects the heterogeneity in the assessment of the heteroscedasticity throughout the studies. Following this suggestion and similar comment of Professor Ian White, we have tried to clarify that mu is a measure of heteroscedasticity and tau is a measure of the heterogeneity of the former throughout all the studies. See also the answer to question 4 for more clarification.9. Supplementary Table S4. Why not put this Table in Section 4, and make one overview of all the models fitted? And I guess that e_ij should be e_i here.Thank you, we have corrected the subscript typo: “e_i”And yes, your suggestion facilitates readability. We have interspersed all the tables and figures of the supplementary material in their respective sections.10. Results: I did not find Figure S1 and Figure S2 very informative. Why not just give a histogram of log(var_OT/var_CT) etc.We have kept Figures S1 and S2 because we believe that they provide additional information about whether or not the increase (or decrease) in the variability in the outcome of the experimental arm depends on the outcome variability of the control arm (or on the baseline variability of the experimental group). However, we have also added the histograms you mention in order to summarize the essential information. The histograms can be seen here or in the Supplementary material.11. Table 1: How were the results from the random model obtained (the 11 increased, 19 decreased etc)?We have obtained them as the studies that had to be removed in order to obtain heterogeneity (i.e., tau) similar to the baseline (which we expect to be null by randomization). We have tried to clarify this point in the legend of the table:“…or (2) number of studies that have to be deleted from the random-effects model in order to achieve a negligible heterogeneity (studies with more extreme outcome were removed one by one until achieving an estimated value of τ similar to the one obtained from the reference model. See Methods section for more details…)”12. Figure 3. Please explain what V_OT, V_OC etc is, as Figures should be self-explained.Thanks. We have included a legend in this figure explaining these abbreviations:V_OT: Variance of the Outcome in the Treated armV_OC: Variance of the Outcome in the Control armV_BT: Variance of the Outcome at baseline in the Treated arm 13. I did not understand the second paragraph of the discussion. I guess that you want to say that the average treatment effect can be interpreted as an individual treatment effect, but I was confused at first by the words “non-observable patient treatment effect”.You are right. We say “non-observable” for the fundamental problem of causal inference (both potential responses are not observable in the same patient), which avoids seeing the treatment effect at the individual level. We have clarified this point:Before: This means that treatment effects obtained by comparing the means between groups can be used to estimate both the averaged treatment effect and the non-observable patient treatment effect.After: This means that the average treatment effect can be interpreted as an individual treatment effect (not directly observable).14. Shocking to see that so many studies do not report measures of variability.Yes. It is really surprising that 61.6% of studies do not report the variability either at baseline or at the end of the study. Although CONSORT advises it, this guideline does not provide the historical data on this practice with which it can be compared.15. The fourth limitation: “the random effect model reveals additional heterogeneity”. To which result are you referring here, comparisons at baseline, followup or over time? The estimate of tau? Why should this be the result of methodological accuracies?We are referring to the main analysis: comparison between arms. Nevertheless, this sentence could be applied to all analyses. Heterogeneity among studies is measured by tau (see response to question 4). We stated that methodological inaccuracies can be derived in the presence of heterogeneity. In an ideal scenario of constant treatment effect in all the studies, the only thing that could lead to heterogeneity in the model would be methodological inaccuracies such as those mentioned in the manuscript or in the referenced paper of Carlisle: transcription errors, insufficient follow-up time for being able to observe this constant effect, or the manipulation of the results in order to achieve greater impact. 16. Figure G is of interest because this is a situation where precision medicine is of interest: for some patients treatment T would be a better choice, for others treatment C and by performing precision medicine the subgroups with different responses could be detected and tailored prescriptions could be given. This indicates that observed homoscedasticity in a study should be interpreted with care and background knowledge of a study is needed to assess whether a situation as in Figure 4 is plausible.Fully agree, although this is a highly sophisticated scenario that we hope will not be viewed as a frequent scenario.Of course, we think that personalized medicine has already been demonstrated to be effective in some areas. Our point is that unless those demonstrations exist, most interventions should be routinely administrated to all patients fulfilling eligibility criteria." } ] } ]	1	https://f1000research.com/articles/7-30
https://f1000research.com/articles/8-842/v1	10 Jun 19	{ "type": "Method Article", "title": "de.NBI Cloud federation through ELIXIR AAI", "authors": [ "Peter Belmann", "Björn Fischer", "Jan Krüger", "Michal Procházka", "Helena Rasche", "Manuel Prinz", "Maximilian Hanussek", "Martin Lang", "Felix Bartusch", "Benjamin Gläßle", "Jens Krüger", "Alfred Pühler", "Alexander Sczyrba", "Björn Fischer", "Jan Krüger", "Michal Procházka", "Helena Rasche", "Manuel Prinz", "Maximilian Hanussek", "Martin Lang", "Felix Bartusch", "Benjamin Gläßle", "Jens Krüger", "Alfred Pühler", "Alexander Sczyrba" ], "abstract": "The academic de.NBI Cloud offers compute resources for life science research in Germany. At the beginning of 2017, de.NBI Cloud started to implement a federated cloud consisting of five compute centers, with the aim of acting as one resource to their users. A federated cloud introduces multiple challenges, such as a central access and project management point, a unified account across all cloud sites and an interchangeable project setup across the federation. In order to implement the federation concept, de.NBI Cloud integrated with the ELIXIR authentication and authorization infrastructure system (ELIXIR AAI) and in particular Perun, the identity and access management system of ELIXIR. The integration solves the mentioned challenges and represents a backbone, connecting five compute centers which are based on OpenStack and a web portal for accessing the federation.This article explains the steps taken and software components implemented for setting up a federated cloud based on the collaboration between de.NBI Cloud and ELIXIR AAI. Furthermore, the setup and components that are described are generic and can therefore be used for other upcoming or existing federated OpenStack clouds in Europe.", "keywords": [ "de.NBI", "de.NBI Cloud", "Cloud Computing", "OpenID Connect", "Life Sciences", "ELIXIR", "Authentication", "Authorization" ], "content": "Introduction\n\nIn life sciences today, the handling, analysis and storage of enormous amounts of data is a challenging issue. For example, new sequencing and imaging technologies result in the generation of large scale genomic and image data. Hence, an appropriate IT infrastructure is necessary to perform analyses with such large datasets and to ensure secure data access and storage. The fully academic de.NBI Cloud, which is operated by the German Network for Bioinformatics Infrastructure (de.NBI), offers a solution to enable integrative analyses for the entire life science community and the efficient use of data in research. To a large extent, de.NBI Cloud will close the gap of missing computational resources for researchers in Germany.\n\nThrough a cloud federation concept, the five de.NBI Cloud sites, located in Bielefeld, Freiburg, Giessen, Heidelberg and Tübingen, are integrated into a single cloud platform, where the central access point of the federation is represented by the web-based de.NBI Cloud portal. The portal is a crucial part of the federated cloud concept and offers an easy entry point for de.NBI Cloud users and an important management tool for cloud and project administrators.\n\nIn collaboration with the European life-sciences Infrastructure for biological Information (ELIXIR) initiative, the de.NBI Cloud portal manages the authorization of users and offers single sign-on to all cloud centers. Perun1, the identity and access management system used in ELIXIR, acts as a backbone for de.NBI Cloud. The interaction between the three entities: portal, cloud site and Perun, as well as the final federation concept that represents de.NBI Cloud, is explained in the following sections. This article demonstrates one way how to build a federated Cloud and the described components and concepts can be reused for upcoming or existing federated clouds.\n\n\nMethods\n\nUser workflow. One of the main goals of de.NBI Cloud is to connect compute centers and the web-based portal through a federation concept. The workflow for a user who wants to start working in the cloud is depicted in Figure 1. The applicant must have an ELIXIR account and be a principal investigator at a German university or research institution. The application is submitted through a web form and is then reviewed by the cloud governance and an access committee. The cloud governance ensures the position of the applicant and determines, in consultation with the access committee, the appropriate resource allocation for a project. Once the project is approved, it can be hosted on one or a multiple of the five compute centers. The principal investigator can add any other user with an ELIXIR account during the project lifetime. Furthermore, they are responsible for any actions of their project members in the cloud.\n\nThis workflow implies multiple challenges, such as easy access to the portal and the centers, authorization of users with different roles and project coordination and configuration. The following sections explain these challenges and how they have been solved by the steps described in Implementation section.\n\nEasy access. The de.NBI Cloud team wants to provide the easiest possible way to access the cloud. As described in the User Workflow section, the user must access the portal and also at least one of the compute centers, which at first glance means that multiple independent accounts must be created for every user. Creating user accounts and passwords means additional work for administrators and users.\n\nUser authorization. De.NBI users can have different roles. For instance, users can be members of a project and, if they are principal investigators (PI), they can be also managers of a project and add other users as members.\n\nFrom the administration perspective, there is the compute center administrator, who maintains the OpenStack installation, and a scientific committee, who must approve PIs, approve projects and assign projects to different compute centers. In addition, de.NBI Cloud must ensure that the current user agreement according to the General Data Protection Regulation (GDPR) is accepted by all users.\n\nProject coordination and configuration. A project can have a multitude of parameters that the user applies for in the de.NBI Cloud portal, which should be mirrored in the specific OpenStack project. Even though all cloud centers are OpenStack-based, they still differ in their version and to a certain extent in their setup, such as the usage of a jump host in one of the centers. In this scenario, the user must first ssh to a dedicated machine and then connect from it to a virtual machine provided by the cloud site.\n\nExample project parameters are number of cores and amount of RAM, but also user specific parameters like an SSH public key, which allows the user to access a virtual machine. This also raises the question of how to transfer the configuration and the permissions of users to a specific OpenStack installation in an automatic way.\n\nOpenStack. OpenStack is an open source Infrastructure as a Service (IaaS) system and allows users to define and start virtual machines. In the context of de.NBI, all cloud sites are operating an OpenStack installation. Keystone is the identity service of OpenStack and provides the authentication and authorization functionality of OpenStack.\n\nPerun. Perun, as described in 1, is a system that provides functionality, covering management of the whole user life cycle in today’s e-Infrastructures, from user enrollment into the e-Infrastructure to user expiration. The Perun system supports management of virtual organizations, rights delegation, group management and enrollment management to provide flexible and easy to use user management. In comparison to ordinary identity management systems, Perun also provides service and access management.\n\nSingle sign-on (SSO). SSO, according to 2, is the ability of a user to authenticate once to a single authentication authority and then access other protected resources without reauthenticating. This concept can be applied to de.NBI Cloud as follows: the authentication authorities are a university, Google, LinkedIn and ORCID. The protected resource is the de.NBI Cloud portal and the cloud sites.\n\nShibboleth. Shibboleth is an identity management system based on SAML3, enabling single sign-on for users. In the context of de.NBI, universities are the identity providers, many of which use Shibboleth. Services like OpenStack installation use Shibboleth to allow users to log in with their home institutional account.\n\nELIXIR Authentication and Authorization Infrastructure (AAI). ELIXIR AAI4 allows researchers to log into services using identity providers available within eduGAIN - the interfederation of identity federation across the globe. Furthermore, ELIXIR AAI offers participating services additional functionality, such as group and attribute management, dataset authorization system or a multi-factor authentication. De.NBI Cloud is fully integrated with ELIXIR AAI. Ideally, the user is able to use his/her university account to access the cloud and any other service provided by de.NBI Cloud. If the university is not part of eduGAIN, alternatives such as Google, LinkedIn or ORCID can be used.\n\nOpenID Connect (OIDC). OpenID Connect, as explained on its website, is one of the SSO protocols and is placed on top of the OAuth 2.0 protocol5. It allows clients like the de.NBI Cloud portal to receive information about end user and thereby to verify their identity based on the authentication performed by an Authentication Server.\n\nThe federation type of de.NBI Cloud comes closest to the definition of an Inter-Cloud Federation Framework (ICFF), as described in 6. In the case of de.NBI, the Inter-Cloud federation broker is represented by the de.NBI Cloud portal, which coordinates and allocates resources.\n\nIn de.NBI Inter-Cloud Federation Framework, the OIDC authorization framework is used for transferring data between the following entities: 1) user agent and the de.NBI Cloud portal, 2) the portal and Perun, 3) user agent and OpenStack.\n\nThe implementation of each step is depicted in Figure 2. Once the user logs into the portal (Step 1), they must be authenticated against an identity provider enabled for ELIXIR AAI and also registered for the de.NBI Cloud virtual organization. By logging into the portal, data, such as information about the identity provider (e.g: university or Google account) and the position of the user in the institution, can be retrieved by the portal. Whereat the position (e.g. employee, member, student) can just be accessed if an identity provider of an university was used.\n\nUsing the token provided by OIDC, the portal can make API calls to Perun and save data, such as de.NBI Cloud-specific project configuration (Step 2). Furthermore, de.NBI Cloud reuses Perun roles and translates them into de.NBI Cloud specific user roles (see Table 1).\n\n1. User logs in to the portal and applies for a project. 2. The Virtual Organisation manager configures the project. 3. Project configuration data is saved in Perun. 4. The project data is propagated to an OpenStack installation.\n\nAll requests are made on behalf of the user, with their own access token (Step 3). The user role in Perun influences the data shown and functionality offered on the portal. In addition, any information about a de.NBI Cloud project can be saved in group-specific attributes in Perun. A number of defined attributes are listed in Table 2. For example, a project manager in de.NBI Cloud can add ELIXIR users to their project because he or she is also group manager in Perun. Each project is assigned programmatically by the portal to a facility whereby a facility in Perun is a compute center in de.NBI Cloud.\n\nAttributes marked with (*) are not yet used by the portal or the PerunKeystoneAdapter.\n\nA facility can propagate its data over HTTPS or SSH (Step 4) as soon as any project-related data changes occur, such as changes to project members or the project quota. The propagated data is in JSON format and consists of project specific configurations, quotas and user-specific SSH keys and ELIXIR IDs. This data does not contain OpenStack-specific information and could be translated into any Infrastructure as a Service System. In the case of de.NBI Cloud, we built an open source tool called PerunKeystoneAdapter, which translates the data into Keystone. It can be extended and customized to the setup of the specific cloud site, such as setting ELIXIR login names on a jump host. Once the ELIXIR ID of a user is saved in Keystone, they can enter OpenStack through Shibboleth or OIDC, depending on the implementation used in the OpenStack setup (Step 5). Finally, the fact that every user must be registered for the de.NBI Cloud virtual organization is a useful administrative feature. If a user agreement is updated, it must be first accepted by a user before they can access any de.NBI Cloud related service (portal, cloud site).\n\n\nConclusions\n\nDe.NBI Cloud represents a federated cloud, allowing users to access the web portal and all five cloud centers with just one account. With the described concept, it is easy to guide the user from the application process until the start of a virtual machine. From the de.NBI Cloud developers perspective, the implementation is made of loosely coupled components (portal, Perun, cloud center) that makes it easy to extend the cloud with additional centers or customize certain components of any cloud site setup. Even the usage of a different Infrastructure as a Service system could be easily implemented without modifying the portal, Perun or any of the existing cloud sites, which allows for the adaption of our concept by other federated clouds. Additionally, administrative features, such as the necessary approval of project applicant positions or ensuring the agreement of users of all cloud sites to the current policy, can be done with minimal effort.\n\nIn summary, ELIXIR AAI represents the backbone of the de.NBI Cloud federation and, in conjunction with OpenID Connect, it enables features that are promising for the future. Two of those features are explained in the Outlook section.\n\nThe concept of OpenID Connect in the context of ELIXIR AAI allows an integration of \"third party\" software and to delegate permissions to them to access certain cloud sites. In the context of de.NBI, it means that de.NBI services can be registered for de.NBI Cloud and, by using OpenID Connect, the service can outsource workloads on demand to the Cloud.\n\nFurthermore, ELIXIR AAI enables the design of a permission API to restrict access to sensitive data, such as human data. A proof of concept is already implemented by the CSC cloud, in which they demonstrate how a virtual machine can access datasets of the European Genome-phenome Archive (EGA).\n\n\nData availability\n\nNo data are associated with this article", "appendix": "Grant information\n\nThis work receives funding from Federal Ministry of Education and Research in Germany (BMBF) [031A537B, 031A533A, 031A538A, 031A533B, 031A535A, 031A537C, 031A534A, 031A532B].\n\nWe acknowledge support for the Article Processing Charge by the Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.\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\nProchazka M, Licehammer S, Matyska L: Perun - modern approach for user and service management. In 2014 IST-Africa Conference Proceedings. IEEE, 2014. Publisher Full Text\n\nDe Clercq J: Single sign-on architectures. In Infrastructure Security. Springer Berlin Heidelberg, 2002; 40–58. Publisher Full Text\n\nRagouzis N, Hughes J, Philpott R, et al.: Security assertion markup language (SAML) v2.0 technical overview. Technical report. 2008. Reference Source\n\nLinden M, Prochazka M, Lappalainen I, et al.: Common ELIXIR Service for Researcher Authentication and Authorisation [version 1; peer review: 3 approved, 1 approved with reservations]. F1000Res. 2018; 7: pii: ELIXIR-1199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHardt D: The OAuth 2.0 Authorization Framework. RFC 6749, RFC Editor, 2012. Reference Source\n\nAssis MRM, Bittencourt LF: A survey on cloud federation architectures: Identifying functional and non-functional properties. J Netw Comput Appl. 2016; 72: 51–71. Publisher Full Text" }	[ { "id": "49755", "date": "02 Jul 2019", "name": "Enol Fernández-del-Castillo", "expertise": [ "Reviewer Expertise cloud computing" ], "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 presents the de.NBI IaaS federation which brings together 5 different OpenStack installations for researchers in life science. The system provides single-sign on by leveraging federated identity technology such as SAML and OpenID Connect with ELIXIR AAI, an established source of identity in the life science community in Europe.\n\nAccess to the federation is managed by a central de.NBI portal that manages users in projects using Perun and synchronises that information to the different OpenStack providers using an Open Source tool. Source code of the portal is not available (or not references in the article at least), but it may be interesting for other similar initiatives to replicate the setup. Authors should consider adding a reference to the source code if available.\n\nDescription of the federation is clear. Comparison with other existing cloud federation initiatives would improve the article.\n\nde.NBI is sometimes written as De.NBI, this should be consistently capitalised.\n\nConsider using the \"Software Tool Article\" instead of \"Method Article\".\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "49751", "date": "02 Jul 2019", "name": "Dixie Baker", "expertise": [ "Reviewer Expertise Security architecture and technology", "particularly as applied for health care and biomedical 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 abstract claims that this paper “explains the steps taken and software components implemented for setting up a federated cloud based on the collaboration between de.NBI Cloud and ELIXIR [Authentication and Authorization Infrastructure (AAI)].” Unfortunately, the paper falls short of its promise. Rather, the paper seems to be an unnecessarily complex and confusing description of both the problem being addressed and the approach taken for the de.NBI.\nFundamental objectives that de.NBI, and others seeking to federate identity and authorization across multiple service providers, need to address are 1) to gain assurance that an individual user is the identity he/she asserts (i.e., user authentication); 2) to obtain reliable information about the security attributes of each authenticated individual (i.e., authorization); and 3) to share an authenticated identity and authorization attributes with other trusted nodes in a network (i.e., identity federation) to the level of assurance required by each node.\n\nAlthough methods and standards used for authenticating identity and sharing security attributes (e.g., SAML, OIDC) differ in ways described in their respective specifications, every method relies on trust established through metadata (e.g., client_id, expiration, scope), and digitally signed (and optionally encrypted) tokens (e.g., JWT, SAML2). The key challenge that de-NBI (and other organizations) is facing is how to achieve syntactic and semantic interoperability among these methods so that data service providers can confidently make access-control decisions based on the tokens they receive from their respective trusted service providers. This paper never succinctly states these objectives and central challenge.\n\nThe “software components” listed include some basic concepts (e.g., “Single sign-on”), some components that are part of other components (e.g., Perun, Elixir AAI), and some components that include services provided by other components (e.g., OpenStack’s Keystone). How these components operate together for de.NBI is not explained. The workflow given in Figure 2 is unclear and confusing – two authentication paths (labelled paths 1 and 5) are shown, both involving Elixir and both including OIDC. It appears that project configuration data are saved only through path 1. The explanation of OIDC “as explained on its website” is superficial at best. Both OpenID Connect (OIDC) and OAuth 2.0 are worthy of more complete explanation.\n\nMany identity brokers capable of providing single sign-on service across SAML and OIDC exist – including the broker used by Elixir itself. I fail to see the value that this paper would provide to its readers.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [] }, { "id": "49754", "date": "15 Jul 2019", "name": "Volker P. Brendel", "expertise": [ "Reviewer Expertise computational genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes the implementation of de.NBI Cloud, which gives integrated access to currently five compute centers through one portal. The end user needs are well stated, and the technical challenges to meet those needs are clearly described. It is outside of my expertise to comment on the technical soundness of the implementation, and no user survey statistics are provided to assess user satisfaction.\nAs de.NBI Cloud is clearly not the first implementation of such a system, I am surprised that the authors don't discuss precedents. For example, I have been a happy user of XSEDE, which is probably the largest similar network. Within XSEDE, Jetstream provides access to virtual machines (run off machines at either TACC or Indiana University). I believe the backend implementation is rather similar, but maybe that should be discussed in the article.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-842
https://f1000research.com/articles/8-823/v1	10 Jun 19	{ "type": "Research Article", "title": "Establishment and phenotyping of neurosphere cultures from primary neuroblastoma samples", "authors": [ "Jack Barton", "Katherine Pacey", "Neha Jain", "Tessa Kasia", "Darren Edwards", "Christine Thevanesan", "Karin Straathof", "Giuseppe Barone", "John Anderson", "Jack Barton", "Katherine Pacey", "Neha Jain", "Tessa Kasia", "Darren Edwards", "Christine Thevanesan", "Karin Straathof", "Giuseppe Barone" ], "abstract": "Background: Primary cell culture using serum free media supplemented with growth factors has been used in a number of cancers to propagate primary cells with stem like properties, which form as spherical cellular aggregates. Methods: We systematically evaluated the capacity of freshly disaggregated neuroblastoma tumors to become established as neurospheres in stem cell media using a uniform protocol. 67 primary neuroblastoma samples from patients treated at a single institution were prospectively evaluated for their ability to become established in culture. Samples, either solid tissue or cells from surgical transit fluid both post chemotherapy and chemotherapy naïve, were evaluated from diagnostic needle biopsies or surgical resections. Results: Overall 37 neurosphere cultures were successfully established from 67 samples. In 11 out of 14 cases investigated by flow cytometry, uniform staining for neuroblastoma markers CD56 and GD2 was demonstrated in CD45 negative non-hemopoietic cells, confirming neuroblastoma origin. Conclusion: We present a simple and reproducible approach for producing primary neurospheres from neuroblastoma samples, which provides a reliable resource for future work including genetic analysis, stem cell research and models for therapeutics.", "keywords": [ "neuroblastoma", "neurosphere", "stem cell" ], "content": "\n\n\n\n\nBackground\n\nNeuroblastoma is the most common extra-cranial solid tumor occurring in children, and the most commonly diagnosed cancer in children under the age of 1 year1. Tumors are thought to have embryological origin in the neural crest, arise within the sympathetic chain or adrenal gland, and are frequently metastatic2. The disease is strikingly heterogeneous with a range of clinical phenotypes, from spontaneous regression, including in cases with metastases, to aggressive disease with marked resistance to radiotherapy and chemotherapy. New approaches to therapies supported by the availability of suitable tumor models that more closely resemble the human disease are essential for improving outcomes for this disease.\n\nEstablished neuroblastoma cell lines have the advantage of being widely available, and enable comparison of research worldwide by their use as a common standard. However, cell lines are limited in their ability to reflect disease physiology due to adaptation to prolonged culturing conditions. Inevitable evolution and changes in transcriptional response occur during culturing, which vary between laboratories3,4, and may alter diverse cell behaviors such as drug response. The potential for proliferation of subclones with greater intrinsic capacity to survive in vitro may lead to loss of genetic variability within a cell line compared with polyclonal cancer populations in humans.\n\nIncreasingly it is recognized that in vivo mouse models of tumors, established in immunodeficient animals following minimal or no in vitro passaging (Patient-Derived Xenografts; PDX) is a valuable tool for evaluation of therapeutic agents. Whilst many PDX models have been established by immediate surgical implantation of freshly acquired tumor surgical samples, this is not always a practical approach. Neurospheres are spherical cellular clusters derived from neural stem cells, that develop under culture conditions of serum free media supplemented with growth factors5. Initially produced from adult CNS tissue, they were the first demonstration of proliferative capacity in the adult brain, and have since been derived from embryonic stem cells, and CNS tumor cells. Cancer stem cells have been identified in a range of malignancies6, and have been implicated in initiation and progression of solid tumors7, and development of resistance to therapy. Growth of neurospheres in stem cell media is hypothesized to enrich for cancer stem cells. Given the importance of cancer stem cells as a potential target for treatment options, and the advantages of primary cultures over standard cell lines, it is necessary to clarify and validate methods for culturing neurospheres from primary material in neuroblastoma.\n\nIt is therefore important to investigate avenues of utilizing shorter term primary cultures such as those established in stem cell media, based on the hypothesis that they may still remain representative of the driver genetic features of the original tumor stem cells, and have limited adaptive changes to tissue culturing conditions. Hence, we sought to establish neurosphere primary lines from neuroblastoma surgical samples, to determine the reproducibility of the technique, and to generate a resource for future research studies in therapeutics, genetics and stem cell biology. We found a high overall success rate (55%) of establishment from surgical samples.\n\n\nMethods\n\nThe study had ethical review board approval (REC reference 14/WM/1253 “Establishing primary cultures and cell lines from pediatric cancers”) and samples were made available following informed consent. Patients were eligible if there was a known or suspected diagnosis of neuroblastoma. All samples were included based on known or suspected diagnosis of neuroblastoma without selection on clinical criteria. In some cases with insufficient material for culture, the transport fluid used for sample transfer (0.9% saline) was placed directly into culture.\n\nTru-cut needle biopsies or tissue from tumor resections from neuroblastoma patients were transferred directly from operating theatre to the hospital histopathology laboratory. Following sterile cut up and routine diagnostic processing including freezing of material for research, surplus material was evaluated by a consultant pathologist for tissue viability and made available for culture. Tissue was manually disaggregated in a 10cm tissue culture dish using a sterile scalpel. Tumors that dissociated readily in this way were placed immediately into stem cell media in 25cm2 flasks, 24 well plates or 12 well plates depending on the available tissue. Since it is not possible to perform an accurate cell count on the partially dissociated tumor, choice of culture container was based on estimation of size that would yield approximately 100% confluence, were the sample fully disaggregated. Where spare solid material was unavailable from a biopsy, up to 1ml of saline used to transport the sample to the histopathology laboratory was taken and added to 4ml of stem cell media in a 25cm2 flask. When more than 1ml of transit fluid was provided, it was centrifuged at 300G for 5 minutes and the pellet resuspended in stem cell media. Samples that were tougher to disaggregate were digested with Accutase (Thermofisher 00-4555-56) for up to 1 hour, or until obvious disaggregation was observed, and divided between wells of a multiwell plate based on the estimation of confluence as above.\n\nDisaggregated cells and tumor fragments were placed into standard serum free neurosphere culture media (Stem Cell Media, SCM) composed of DMEM/F12 (Sigma-Aldrich D8437),° 1% B27 (Gibco 17504044) with 20ng/ml EGF (Sigma Aldrich #E9644) and 20ng/ml FGF (Fibroblast Growth Factor, Peprotech 100 – 18B) in the presence of penicillin and streptomycin antibiotics. Cells were propagated at 37°C in 5% CO2. Cultures were inspected twice a week for cell density and presence of neurospheres, and split 1:2 to 1:5 depending on cell density and speed of growth. Cultures were frozen in serum free DMEM/F12 media with 10% DMSO at approximately 107 cells per ml.\n\nA total of 14 randomly selected representative primary cultures were analyzed by flow cytometry. To produce a single cell suspension necessary for this, cells growing as a monolayer were detached with Accutase (Thermofisher) according to the manufacturer’s instruction, while neurospheres were disaggregated with Accutase and the mechanical force of gentle pipetting. Surviving cells, re-suspended into single cell suspension, were stained in FACS tubes using saturating amounts (1–5ul of stock) of antibody in 100ul PBS using the following panel of directly conjugated monoclonal antibodies all from Biolegend: GD2-PE (357304, RRID: AB_2561885), CD56-APC/Cy7 (318332, RRID: AB_10896424), CD45-BV711 (304049, RRID: AB_2563465) controlled for nonspecific staining using isotype-matched labelled polyclonal antibodies. The stained primary culture cells were incubated on ice with antibody mix for 30 minutes and then washed with PBS and centrifuged at 300G for 5 minutes. Data was collected with the BD LSRII flow cytometer (BD Biosciences) and data analysis used FlowJo software (v8.8.3).\n\nThe gating strategy excluded dead cells by a live/dead stain using DAPI. To exclude leukocytes that may be present from blood in the original sample, the resulting live population was gated on CD45 negative and CD56 positive cells, the latter a standard marker for neuroblastoma. This gated cell population of CD45-ve/CD56+ve cells was then examined for expression of GD2, also ubiquitously expressed on neuroblastoma.\n\nChi-squared tests were used to determine significance of association of categorical variables; for example successful versus non successful expansion correlated with pre versus post chemotherapy. Data were tabulated and tests of significance were performed using Microsoft Excel 2016 and GraphPad Prism v6.0\n\n\nResults\n\nBetween October 2014 to end of 2016, tumor biopsies or resections of 67 consecutive neuroblastoma samples from 52 patients treated at Great Ormond Street Hospital London were systematically evaluated for their ability to establish primary neurosphere lines. Of these, 39 were primary needle biopsies and 28 were surgical resections of which 24 were post chemotherapy resections and 4 were primary (treatment-naïve) resections.\n\nThe success in terms of establishment of cultures was determined by the ability of cultures to produce discrete visible neurospheres. All cultures derived from neuroblastoma samples were observed to grow as a mixture of phenotypes including neurospheres, single suspension cells, and as monolayer, although often one of the three growth patterns was predominant. Cases could be dichotomized into those that formed spheres within the first 14 days of establishment and then went on to form long term successful cultures, and those that never formed spheres and were classified as “unsuccessful cultures”. The overall success rate, as thus defined, was 55%. It appeared that successful establishment of neurospheres could be made from chemotherapy-naïve primary tumors and post chemotherapy surgical samples, as well as primary needle biopsies. Successful establishment of lines is summarized in Table 1. Of 43 chemotherapy-naive samples (39 biopsies and 4 surgical excisions), 29 (67%) were successfully established, whereas for the post-chemotherapy surgical samples success rate was 8 out of 24 (33%), which is significantly inferior (Chi Sq p=<0.008) (see underlying data8). This suggests that the protocol, if applied to optimally procured tissue, would have a success rate over 65%.\n\nOf the chemotherapy-naïve surgical excisions, 2 of these 4 were from recurrences.\n\nDue to the impossibility of counting cells both at the start of culturing and following establishment of spheres, it is not possible to plot growth curves. However, the range of time from initial seeding to establishment of a confluent 75cm2 flask is approximately 2 to 10 weeks showing the marked heterogeneity of growth rates.\n\nIn order to determine that these were neuroblastoma cells, 14 representative samples were analyzed by flow cytometry. Cells from primary cultures were assessed for expression of the pan leucocyte marker CD45, which neuroblastoma cells do not express, and for ubiquitously expressed NBL markers CD56 and GD2. Of the samples tested, 79% exhibited this characteristic neuroblastoma staining pattern. We found that the neurosphere cultures showed a very bright and homogeneous staining for both NBL markers (Figure 1)\n\nA) representative flow cytometric staining for two independent cultures (excision culture 3544 (upper) and biopsy culture 4669 (lower) following gating on live cells. B) graphical representation of the staining pattern in 14 evaluated samples; positivity was categorized into dim, medium and bright based on MFI and scored as 1, 2 and 3 respectively.\n\nWe were interested in whether it is possible to establish neuroblastoma lines when using very small amounts of tissue. In cases with insufficient tissue to culture, 1ml of transportation fluid was put directly into 4ml stem cell media (SCM) in a 25cm2 tissue culture flask, testing the hypothesis that small amounts of residual tumor cells within the transport media would be able to establish. Where transport fluid provided was more than 1ml, this was centrifuged and the pellet resuspended in SCM. Cultures established from transit fluid had a comparable success rate to those established from solid tissue samples (9 out of 15= 55% versus 25 out of 39 =64% for biopsies: Chi Sq p=0.78).\n\nTo perform a further unbiased comparison, eight patients had neurospheres cultured from the same biopsy with separate culturing of the solid tissue and surgical transit fluid. In three there was no successful growth, in three there was concordance of growth from the two sources, and in two there was success from solid tissue but not transit fluid; there were no cases with success of transit fluid but failure from solid tissue. Therefore transit fluid appears a less reliable source of material but a useful adjunct in the context of scarce material.\n\nIn addition to growth as neurospheres, primary cultures established in SCM were observed to grow as adherent monolayers, single cell suspensions, or mixtures of all three growth patterns. Moreover, there is marked heterogeneity in the size of neuropheres both within and between samples. It is not possible to quantify this heterogeneity, but all the NBL SCM lines contained some elements of adherent, single cell suspension and neurospheres. SCM derived from gliomas can be grown as adherent cultures by coating culture flasks with laminin and so we were interested in whether neuroblastoma primary cultures could also adopt this phenotype. We did not attempt initial establishment in laminin in any cases. In our hands, when established or establishing SCM lines were transferred to laminin, the degree of attachment increased markedly, with outgrowth of adherent spheres and associated adherent cells growing out as a monolayer. In some cases where growth in suspension appeared slow, the change to laminin appeared to accelerate growth. Because of the impossibility of counting cells in neuropheres without majorly disrupting them, it was not possibly to quantify these aspects of altered growth in laminin. On return of laminin cultured cells to non-coated flasks, they reverted to predominant suspension growth pattern.\n\nPatient characteristics and ability to establish neurospheres is shown in Table 2. The number of male samples were 37 out of 67. The ability to establish neurospheres did not vary depending on gender (59% vs 50%). Biopsies which demonstrated only undifferentiated neuroblastoma had a higher but non-significant success rate in establishing neurospheres than those which showed any evidence of differentiation or post chemotherapy changes (60% vs 45%). Age, stage, MYCN amplification and the presence of segmental chromosomal aberrations were all non significant in terms of neurosphere success rate (Table 2).\n\nThere were no significant associations with neurosphere establishment (Chi-square). L1, L2, M or MS stage disease at diagnosis used the International Neuroblastoma Risk Group (INRG) Classification System.\n\nSix patients had two samples collected at different time points during treatment. For four of them there was no concordance for success between the timepoints whereas two patients (one success and one failure) the different timepoints were concordant. Therefore we failed to find any evidence that patient-specific factors govern success rate.\n\n\nDiscussion\n\nHere we present data from a series of 67 samples diagnosed with neuroblastoma, from which tumor tissue or surgical transit fluid was obtained and used to culture tumor spheres under conditions commonly used in the culture of neural stem cells6. Tumor spheres were subsequently dissociated and assessed by flow cytometry for presence of neuroblastoma markers GD2 and CD56, to show that cells and tumor spheres grown in culture retain the characteristic markers the cells of their tissue of origin. These cells may then be used as targets in vitro for drug development, or for engraftment in vivo for the development of patient-derived xenograft models that better reflect the heterogeneity of this disease.\n\nAlthough well established that it is possible to use SCM to propagate stem cells in the form of neurospheres from a number of different cancer types9, the literature is sparse in regards to their evaluation in neuroblastoma. We have therefore systematically evaluated success rate and shown that with simple culturing techniques it is possible to establish primary lines which retain the characteristic immunophenotypic features of neuroblastic tumors. Further studies will be required to establish the success rate of formation of tumors, for example in xenograft models. Moreover, it will be interesting to study the ability of these cells to differentiate into neuroblasts in vitro, for example by transfer to normal growth media with serum, as well as the mutational drift compared to the original tumor if any.\n\nIt would be advantageous to have a method of establishment of primary cell culture which reproducibly leads to cell expansion. Even if the expanded cells represent a subclone that are adapted to in vitro conditions (for example cancer stem cell expansion in stem cell media), this still represents an advantage over methods such as establishment of cell lines in normal growth media with serum, that typically go through a crisis phase before establishment of an emergent line that is presumably highly evolved for adaptation to tissue culture conditions, and therefore less likely to represent the in vivo tumor. Conventional two-dimensional monolayer models have been useful in understanding many of the characteristics of neuroblastoma cell biology, but in addition to the concerns about genetic evolution to adapt to typical cell culture conditions, they do not accurately reflect the multicellular physiology of tumors in vivo or how the tumor microenvironment is developed. Neither adherent monolayer cultures nor neurospheres are physiological, and occur only during in vitro culturing conditions, however the latter may offer some advantages as a research tool. Although neurospheres do not have the architecture and vasculature of in vivo tumors, their three-dimensional structure may have advantages of cell monolayers in terms of better representing aspects of three-dimensional tumors such as central hypoxia and drug penetrance. Future studies on neurosphere permeability and response to therapeutics might be of interest to evaluate their role as an in vitro tumor model.\n\nThe establishment of neurospheres comprised of primary neural stem cells has also been frequently used and there exist a variety of methods for their establishment6. However, the sphere culture model is limited by its sensitivity to culture technique including sensitivity to alterations in media or cell density, or frequent passaging and dissociation5,10. In our hands the neuroblastoma SCM cultures do show this sensitivity to changes in environmental conditions but retain the capacity to recover from stress if left alone in normal SCM growth conditions. Due to the long-term nature of their propagation, meticulous attention to sterility is needed to prevent the outbreak of microbial contamination.\n\nWhile neurospheres represent a straightforward approach for maintaining cancer cells in culture, and whilst they are a potentially useful tool for cancer stem cell investigations, further studies are needed to determine how useful they will be in developing biological or therapeutics models for basic or translational research. Our demonstration of high success rate of their establishment using a uniformly applied protocol is a valuable contribution to the field for identifying an alternate and reproducible approach to expanding the reagents available for neuroblastoma research.\n\n\nEthics approval and consent to participate\n\nThe study had ethical review board approval (REC reference 14/WM/1253 “Establishing primary cultures and cell lines from pediatric cancers”) and all patient samples were collected with written full consent\n\n\nData availability\n\nOpen Science Framework: Primary neuroblastoma cultures database. https://doi.org/10.17605/OSF.IO/T2RFB8\n\nThis project contains the following underlying data:\n\nFACS data (folder containing output FACS files for different cases)\n\nFlow Cases - reference table.docx (Reference table for FACS output files)\n\nFull data sheet no identifiers.xlsx (spreadsheet of sample characteristics)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis work was supported by the Great Ormond Street Hospital Charity [Leadership award to JA and W1134, VS0118, W1029 and W1076], GOSH NIHR Biomedical Research Centre, Research in Childhood Cancer, Great Ormond Street Hospital Histopathology Department, and the Wellcome Trust through a Clinician Scientist Fellowship.\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\nMaris JM: Recent advances in neuroblastoma. N Engl J Med. 2010; 362(23): 2202–2211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarshall GM, Carter DR, Cheung BB, et al.: The prenatal origins of cancer. Nat Rev Cancer. 2014; 14(4): 277–289. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBen-David U, Siranosian B, Ha G, et al.: Genetic and transcriptional evolution alters cancer cell line drug response. Nature. 2018; 560(7718): 325–330. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPan C, Kumar C, Bohl S, et al.: Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol Cell Proteomics. 2009; 8(3): 443–450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJensen JB, Parmar M: Strengths and limitations of the neurosphere culture system. Mol Neurobiol. 2006; 34(3): 153–161. PubMed Abstract \| Publisher Full Text\n\nClarke MF, Hass AT: Cancer Stem Cells - Clarke - - Major Reference Works - Wiley Online Library. Accessed October 26, 2018. Publisher Full Text\n\nVisvader JE, Lindeman GJ: Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008; 8(10): 755–768. PubMed Abstract \| Publisher Full Text\n\nANDERSON J: Primary neuroblastoma cultures database. 2019. http://www.doi.org/10.17605/OSF.IO/T2RFB\n\nSingh SK, Clarke ID, Terasaki M, et al.: Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003; 63(18): 5821–5828. PubMed Abstract\n\nSingec I, Knoth R, Meyer RP, et al.: Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods. 2006; 3(10): 801–806. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49686", "date": "01 Jul 2019", "name": "Dermot Murphy", "expertise": [ "Reviewer Expertise Neuroblastoma and children's cancer in general. Palliative care", "statistics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is scientifically sound. My only criticism is stylistic. I think the sentences are too long and the punctuation could be better. The text is clunky.\nFor example:\n“The disease is strikingly heterogeneous with a range of clinical phenotypes, from spontaneous regression, including in cases with metastases, to aggressive disease with marked resistance to radiotherapy and chemotherapy. New approaches to therapies supported by the availability of suitable tumor models that more closely resemble the human disease are essential for improving outcomes for this disease.”\n\nEdit to:\nNeuroblastoma is strikingly heterogeneous. Clinical phenotypes range from spontaneous regression to aggressive disease characterised by marked resistance to radio and chemotherapy. New therapeutic approaches, supported by tumor models more closely resembling human disease, are essential for improving outcomes in children.\nThere are many more examples in the subsequent manuscript. Tight editing would vastly improve readability.\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": "49685", "date": "01 Jul 2019", "name": "Toby Trahair", "expertise": [ "Reviewer Expertise Paediatric Haematologist & Oncologist", "translational research in neuroblastoma & personalised 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\nThis is a straightforward technical report on direct culture of neuroblastoma samples from consecutive cases treated at Great Ormond Street.\nThe research team has taken a logical approach to establishing an easy method to establish patient-derived neurospheres. Tumour samples were derived from a number of sources (open biopsy, needle biopsy & from transport fluid). The overall success rate (37 cultures from 67 samples) confirms that this approach is feasible.\nThe characterisation of the resulting neurosphere cultures has been limited, but expression of both CD56 and GD2 suggests that the cultures really are neuroblastic tumours.\nThe significance of this work really lies in the downstream application of patient-derived cultures. There are a number of unanswered questions, specifically how representative the neurosphere cultures are of the donor tumour, whether there is any correlation between the capacity to establish a culture and patient outcome, whether the neurosphere models can be propagated as xenografts, and if it is possible to derive neurosphere cultures from each genetic/genomic subtype of neuroblastoma (e.g. MYCN amplified, TERT rearranged, ATRX deleted, etc.).\nOverall, with an increasing focus on personalisation of therapy, whilst a simple piece of work, I believe it is a valuable contribution to the field and will be helpful for other groups engaged in such work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "49683", "date": "02 Jul 2019", "name": "Juliet C. Gray", "expertise": [ "Reviewer Expertise Neuroblastoma" ], "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\nNeuroblastoma is a rare paediatric tumour which accounts for a disproportionately high number of paediatric cancer deaths. Fresh tissue samples are usually small and scarce, and established cell lines are likely to poorly represent the heterogenous nature of the disease. This paper describes a simple methodology for establishing neurosphere cultures from small samples of fresh biopsy tissue, with a relatively high success rate. This is a potentially very useful technique for extending the use of limited primary tissue samples to maximal benefit. Of note, success was even achieved with fluid from around surgical/biopsy samples, rather than solid tissue samples, which is potentially very useful when sample size is limited.\nThe article is clearly written and will be of interest to those working 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": [] }, { "id": "49684", "date": "08 Jul 2019", "name": "Gudrun Schleiermacher", "expertise": [ "Reviewer Expertise Pediatric oncologist (physician-scientist)", "translational research focusing on genetic analyses of neuroblastoma and other high risk pediatric cancers" ], "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\nNeuroblastoma is a clinically and genetically heterogeneous pediatric tumor. Precise biological and molecular characterization of tumor cells is increasingly becoming an integral part of patient care, with treatment stratification depending not only on clinical, but also on molecular characteristics. However, tumor samples are frequently small, especially when obtained by fine needle biopsies, with frequently only a limited amount of biological material available for increasingly complex molecular investigations or cellular screening procedures.\nIn this manuscript, the authors describe the technical aspects of direct culture of neuroblastoma samples for the establishment of neurosphere cultures. Importantly, they describe a procedure which is applied to consecutive cases in a single center, with samples obtained by different techniques (surgical resections, fine needle biopsies, transport fluid) and at different time points (at diagnosis, post chemotherapy). The success rate is 55% (37/67 samples), without any significant association between the success rate of establishing a neurosphere culture and clinical/histological/genetic features. Characterization of the obtained neurosphere cultures is still limited, but the results indicate that the cultured cells do express neuroblastoma markers.\nThe ultimate usefulness of the established neurosphere cultures will depend on a number of aspects. It will be necessary to compare genetic/epigenetic/cell identity features between the primary tumor and corresponding neurosphere culture, to search more in detail for features linked to culture success rates and to establish the duration of tumor growth.\nThe importance of this work is linked to the presentation of a reproducible method for the expansion of frequently rare tumor tissue, enabling more large scale use of such samples for molecular characterization, drug screening and other applications for the patients’ benefit.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-823
https://f1000research.com/articles/7-1837/v1	22 Nov 18	{ "type": "Software Tool Article", "title": "The InterMine Android app: Cross-organism genomic data in your pocket", "authors": [ "Daria Komkova", "Rachel Lyne", "Julie Sullivan", "Yo Yehudi", "Gos Micklem", "Daria Komkova", "Rachel Lyne", "Julie Sullivan", "Yo Yehudi" ], "abstract": "InterMine is a data integration and analysis software system that has been used to create both inter-connected and stand-alone biological databases for the analysis of large and complex biological data sets. Together, the InterMine databases provide access to extensive data across multiple organisms. To provide more convenient access to these data from Android mobile devices, we have developed the InterMine app, an application that can be run on any Android mobile phone or tablet. The InterMine app provides a single interface for data access, search and exploration of the InterMine databases. It can be used to retrieve information on genes and gene lists, and their relatives across species. Simple searches can be used to access a range of data about a specific gene, while links to the InterMine databases provide access to more detailed report pages and gene list analysis tools. The InterMine app thus facilitates rapid exploration of genes across multiple organisms and kinds of data.", "keywords": [ "Android app", "Genomics data", "Gene search", "InterMine" ], "content": "Introduction\n\nInterMine is a data integration and analysis software system that has been used to create a suite of both inter-connected and stand-alone biological databases for the analysis of large and complex biological data sets. InterMine databases have been developed for the major model organisms budding yeast1, nematode worm, fruit fly2, zebrafish, mouse3 and rat4, (which we will refer to as the Model Organism Database (MOD-) InterMines, together with a human database and databases for plants, bees and wasps5, cows6, Medicago truncatula7, mitochondrial proteomics8 and drug targets9 (Table 1; https://registry.intermine.org/). Together, the InterMine databases provide access to extensive data across multiple organisms (for full listings of data included see the website for each individual InterMine, Table 1). To provide more convenient access to these data from Android mobile devices, we have developed the InterMine app10, an application that can be run on any Android mobile phone or tablet.\n\nThe list can be configured in the app.\n\nThe InterMine app provides a single interface for data access, search and exploration of the above databases. It can be used to retrieve information on genes and gene lists, and their relatives across species. Simple searches can be used to access a range of data about a specific gene, while links to the InterMine databases provide access to more detailed report pages and gene list analysis tools.\n\nThe app is available from the Google Play Store at https://play.google.com/store/apps/details?id=org.intermine.app.\n\n\nMethods\n\nThe InterMine app allows users to search a default subset of InterMine data warehouses (Table 1). This list is configurable, and so users are able to refine or add mines to match their interests. See https://registry.intermine.org for the full list of known public InterMine instances.\n\nInterMine databases typically integrate data from many resources. For instance BioGRID11, IntAct12, UniProt13, and can include high quality curated data (from the Model Organism Databases), genome-wide high-throughput data and data from smaller more focused studies (See individual InterMine websites for more details).\n\nThe InterMine app provides several ways to search and explore the data available, including a keyword search, sets of pre-defined template searches and list analysis functionality. These features are described in more detail in the use case section.\n\nInterMine databases allow users to create an account through which they can, between sessions, store lists and searches. The InterMine App therefore allows users to log in to any accounts they hold on the underlying databases, so enabling user-created lists to be accessed.\n\nUsers are also able to mark genes in search results as favourite. These genes are stored on the Android device and can be accessed without needing to log in to any of the underlying databases.\n\n\nImplementation\n\nThe InterMine App draws all data from the RESTful Application Programming Interfaces (APIs) that InterMine databases provide14. RoboSpice, an Open Source (Apache 2.0 Licence) Android communications library15, provides core network communication functionality. Data are loaded asynchronously over HTTP or HTTPS, depending on the preferred protocol of the database being accessed. For performance enhancement, most web service responses are stored on the device if headers state an appropriate cache lifetime.\n\nWhen the app receives a JSON response from the web service, it transforms the data from a table-structured format to a more hierarchical view, which presents data more effectively on smaller-screened mobile devices.\n\nEach InterMine database is discrete and often maintained by different organisations. If a user wishes to authenticate with multiple InterMines - perhaps to view private gene lists stored on different databases - they will need to provide separate authentication details for each InterMine database. However this is only necessary once, as after a user has successfully authenticated in a given InterMine via a username/password pair, the app retrieves and stores an API authentication token. This ensures that the user can authenticate in the future without having to re-enter or store sensitive password details.\n\nAll of the user configuration settings and authentication tokens are stored locally on the device via SharedPreferences, Android’s dedicated settings storage mechanism16.\n\nTabular data, such as favourite genes within the app, are not suited to the key/value pair storage used in SharedPreferences17, and therefore are stored within an SQLite database on the user’s device. Data stored include the InterMine instance the data originated from, the (e.g.) gene’s identifiers, description, organism, and genomic coordinates.\n\nKeyword search is available across lists, templates and gene search results. Search results from different databases are presented to the user as a single result set, sorted by the search relevance score generated by each originating database. Search results can be shared via email, instant messaging, and other sharing media in text format, using Android’s ACTION_SEND Intent functionality18.\n\nInterMine also includes advanced analysis tools - particularly data visualisations - which may not be available via the API. To access the extended information about genes or gene lists, a user can load InterMine’s advanced report pages within the app itself. This is implemented via Android’s WebView19 functionality which allows live web pages to be embedded in an application (for example, Figure 2 shows an example of an embedded InterMine WebView).\n\n\nOperation\n\nThe app is implemented in accordance with Google Material Design20 guidelines, providing a predictable environment for the user, and also supports Android version 4.0 and above, ensuring it is able to run on over 99% of active Android phones as of November 201821.\n\n\nUse case\n\nThe keyword search simultaneously searches all InterMine databases selected through the settings option. Thus, a cross-organism overview of data available for further investigation is provided. Link-outs from the search results to each originating InterMine database provide access to detailed gene report pages. These pages collate information integrated for that gene and typically include functional summaries, ontology annotation, pathway, expression, interaction and disease data and links to additional related data.\n\nAs an example, searching for ‘dopamine’ returns dopamine-related genes from PhytoMine, MouseMine, HumanMine, TargetMine, FlyMine, RatMine, ZebrafishMine, WormMine, YeastMine, ThaleMine and HymenoperaMine (Figure 1; see Table 1 for urls). Selecting a gene from the results, for instance the human gene DRD4 (dopamine receptor D4) displays summary information about the gene, with a link to the full gene report available from the HumanMine database. Here we learn, for example, that polymorphisms in the DRD4 gene are associated with the disorder attention deficit hyperactivity disorder (ADHD) (Figure 2), a condition associated with low dopamine levels. The search results therefore facilitate rapid exploration across multiple organisms and kinds of data.\n\nA search for dopamine returns genes from PhytoMine, MouseMine, HumanMine, FlyMine, TargetMine, RatMine, ZebrafishMine, YeastMine and ThaleMine. A section of the results from HumanMine and FlyMine is shown.\n\n(a) This view shows the first part of the report page with a summary of the Gene identifiers and data available. (b) This view shows the Disease section of the report page.\n\nIn addition to cross-organism gene search, the InterMine databases provide libraries of pre-built searches, called template searches. Such searches provide a user-friendly interface where the parameters for search filters can be specified. These templates range from simple searches, such as for a specified gene (or genes), return the corresponding Gene Ontology terms (represented as “Gene → Gene Ontology terms”), to more complex searches combining data of more than one type, such as “Tissue + interaction → genes”, which returns any genes expressed in the specified tissue that also interact with the product of a specified gene. Templates from each InterMine database are available within the InterMine App. The results are provided with a simple keyword search to facilitate further data browsing. For instance, continuing the above dopamine example, we can use a template search to identify genes in Drosophila associated with ADHD: on the templates page for the FlyMine database, we find the template “Disease -> Human genes and Orthologues” (Figure 3). This template allows one to specify disease names that contains “attention deficit”, and on running the template, this returns the disease Attention deficit-hyperactivity disorder along with associated human genes and their orthologues from the available InterMine databases. Using the ability to search within the results we are able to verify that the human gene we are interested in (DRD4) is associated with this condition, and that this gene has a predicted orthologue in Drosophila melanogaster, FBgn0053517, Dop2R. Through such iterative searching we can continue our investigation of this fly orthologue to identify, for instance, interacting partners, pathway and Gene Ontology annotations.\n\nThe disease name is an editable constraint, in this case set to search for human genes associated with diseases with a name that “contains” Attention deficit, as well as their orthologues.\n\nShows the human dopamine receptor D4 associated with the disease attention deficit-hyperactivity disorder and with the D. melanogaster orthologue FBgn0053517, Dop2R.\n\nInterMine databases are especially suited to the analysis of lists of genes or other entities. Users can create their own lists, which can be stored between sessions if the user has an account for the relevant InterMine database. Again, direct links from lists to the underlying InterMine databases provide access to list analysis tools, for instance enrichment statistics that help identify surprising properties, such as publications that cite an unexpectedly large number of the list members, or GO terms or protein domains that are associated with an unexpectedly large number of list members.\n\nPublic lists, which are typically interesting sets of genes derived from publications and other studies, are often provided by the database operators. For instance, in FlyMine, eleven of the public lists provide sets of genes whose expression increases at defined times during drosophila embryogenesis, as derived from Hooper et al22. Further lists show genes that are expressed at increased levels in various adult fly tissues according to data from the FlyAtlas resource23. Within one of these sets, PL FlyAtlas_brain_top, we can identify a set of genes up-regulated in the brain. Checking within this list, we find that the dopamine receptor gene Dop2R (FBgn0053517) identified above is present. By following the link to the corresponding list analysis page on the underlying FlyMine website, and examining the enrichment statistics, we find that the Dop2R gene is part of a set in which the Gene Ontology term dopamine receptor signaling pathway (GO:0007212) occurs unexpectedly frequently (p-value 0.001303, with Holm-Bonferroni correction). It is also apparent that two other fly dopamine receptors, Dop1R1 (FBgn0011582) and Dop1R2 (FBgn0266137) are also found in this list (Figure 5).\n\nA set of three genes (FBgn0011582 (Dpo1R1), FBgn0053517 (dop2R) and FBgn0266137 (Dop1R2) are enriched for the GO term dopamine receptor signaling pathway.\n\n\nConclusions\n\nThe InterMine app provides a convenient way of searching for biological information across many model organism and other databases, allowing an overview of gene function and gene relationships to be pursued. Importantly, the InterMine app reduces the effort required to obtain data available in a range of InterMine databases by removing the need to visit each one individually. Further development of the app is planned, including a single sign-in for all of the InterMine instances through oAuth2; further search and analysis capabilities including extending the keyword search to include all data types (instead of just genes); better cross-InterMine search result ordering; an offline mode with data cached in a local database for access when no internet connection is available, and a more sophisticated query construction capability for more advanced users.\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\nThe InterMine app is available from the Google Play Store: https://play.google.com/store/apps/details?id=org.intermine.app.\n\nSource code available from: https://github.com/intermine/intermine-android.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.147864610.\n\nLicense: GNU General Public License v2.", "appendix": "Grant information\n\nThis work was supported by the Wellcome Trust (Grant number: 099133).\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\nBalakrishnan R, Park J, Karra K, et al.: YeastMine--an integrated data warehouse for Saccharomyces cerevisiae data as a multipurpose tool-kit. 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Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1478646\n\nChatr-Aryamontri A, Breitkreutz BJ, Oughtred R, et al.: The BioGRID interaction database: 2015 update. Nucleic Acids Res. 2015; 43(Database issue): D470–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerrien S, Aranda B, Breuza L, et al.: The IntAct molecular interaction database in 2012. Nucleic Acids Res. 2012; 40(Database issue): D841–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015; 43(Database issue): D204–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalderimis A, Lyne R, Butano D, et al.: InterMine: extensive web services for modern biology. Nucleic Acids Res. 2014; 42(Web Server issue): W468–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nstephanenicolas: stephanenicolas/robospice. GitHub. (Accessed: 10th November 2016). Reference Source\n\nSaving Key-Value Sets \| Android Developers. (Accessed: 10th November 2016). Reference Source\n\nSaving Data in SQL Databases \| Android Developers. (Accessed: 10th November 2016). Reference Source\n\nSending Simple Data to Other Apps \| Android Developers. (Accessed: 10th November 2016). Reference Source\n\nBuilding Web Apps in WebView \| Android Developers. (Accessed: 10th November 2016). Reference Source\n\nMaterial Design for Android \| Android Developers. (Accessed: 10th November 2016). Reference Source\n\nDashboards \| Android Developers. (Accessed: 9th November 2016). Reference Source\n\nHooper SD, Boué S, Krause R, et al.: Identification of tightly regulated groups of genes during Drosophila melanogaster embryogenesis. Mol Syst Biol. 2007; 3: 72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson SW, Herzyk P, Dow JA, et al.: FlyAtlas: database of gene expression in the tissues of Drosophila melanogaster. Nucleic Acids Res. 2013; 41(Database issue): D744–50. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "42072", "date": "10 Jan 2019", "name": "Johan Nyström-Persson", "expertise": [ "Reviewer Expertise Bioinformatics", "software development", "genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe the InterMine Android app, which makes data available from InterMine applications on Android devices. Significantly, it is able to combine data from multiple data warehouses, and users can add their own InterMine instances to this list.\nThe source code is GPL licensed and available on GitHub, and the source code as it was at the time of publication has also been archived.\nFor this review, unfortunately I did not have access to an Android device, so I assume that the application functions as described.\nThe app seems to contain a carefully chosen set of core features to enable quick searches, for example by gene or template, and presents combined search results in a streamlined user interface. It is also possible to access gene lists. The WebView feature to display visualisations should also be useful, although most likely the user experience here is harder to control by nature (owing to the different widgets that may be available in InterMine instances being accessed).\nI am curious as to how the app would behave if one queries different InterMine instances that have very different response times and displays the combined results. Hopefully, the app would display results incrementally as they are available, instead of waiting for the slowest instance to reply and then combining the results.\nThe authors have also taken reasonable care to handle authentication details and transmitted information securely.\nIn summary, the InterMine Android App should be useful for querying InterMine instances on the go from handheld devices, but also for combining data from different instances in a way that, as far as I know, has not been possible until now. I recommend acceptance of this report.\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": "43497", "date": "18 Feb 2019", "name": "Zeeshan Ahmed", "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\nAuthors have produced a smart phone app, and I would like to congratulate them for good work.\nHere, I provide some comments:\nIntroduction is brief; there is no background and rational provided, which can justify the need of such application. Moreover, there are no details provided of similar smart phone applications and other related platforms (e.g. web, desktop etc.). I would suggest to address these points comprehensively and add comparative analysis of their app with other related applications, including a table based on common and variable features would be helpful.\nAs authors are interested in publishing their app as a scientific contribution, it’s important to have scientific justifications and discussion. At this time paper is more like a brief report.\nMethodology; why Android based smart phone app, why not iOS?\nIs data behind the app (collection of databases) freely accessible to the users, so they can download, and even verify the results and with other referenced databases? If not, then mention it in the paper, and give reasons for that. As this is an open source work, and data is collected from multiple open sources, its expected to have access to the data linked at the backend.\nAuthors have mentioned list of databases, it’s important to mention licensing information of those database, to avoid any conflict of interest. Moreover, its important to clearly mention it in the conflict of interest section.\nAuthor’s contribution are missing, it’s also important to list those.\nI would suggest to write supplementary material and there explain the app in detail (step-by-step). Guide a new user as to how to get access to the app and how each interface can be used, and what are expected inputs/outputs. Furthermore, if word count restriction does not allow, then further extend supplementary material and provide comprehensive details of software implementation, database design and data workflow with rational for choosing those options. Make some diagrams (based on software engineering concepts) explaining design and implementation parts.\nI would suggest authors to also mention:\nCurrent limitations of the app. Current advantages of the app, which signifies it technically and scientifically. What were the major technical, non-technical, and scientific difficulties they have faced while designing and developing this app. Future recommendations, in their view and for other readers.\nRegarding Figures; Figure 1 seems isolated. I would rather suggest make one good multi panel Figure, and add 1, 2, 3, 4 to that.\n\nIs the rationale for developing the new software tool clearly explained? No\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? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "4661", "date": "07 Jun 2019", "name": "Rachel Lyne", "role": "Author Response", "response": "Authors have produced a smart phone app, and I would like to congratulate them for good work.We thank the reviewer for his detailed comments on the manuscript. We have made several changes to the manuscript and have made further comments below which we hope address the points made:Here, I provide some comments:Introduction is brief; there is no background and rational provided, which can justify the need of such application. Moreover, there are no details provided of similar smart phone applications and other related platforms (e.g. web, desktop etc.). I would suggest to address these points comprehensively and add comparative analysis of their app with other related applications, including a table based on common and variable features would be helpful.As authors are interested in publishing their app as a scientific contribution, it’s important to have scientific justifications and discussion. At this time paper is more like a brief report.As the development of mobile apps for biology is still in its infancy and as this provided a new and novel way of accessing the data in InterMine, it’s development was largely exploratory as we did not know at the outset how much demand for the app there would be. The very popular iOS app developed by the Saccharomyces Genome database (utilizing their yeastMine webservices) suggested that this could become a popular way in which data could be accessed (Wong et al; PMID: 23396302). Indeed, the InterMine app has already had well over one hundred downloads and we hope that with continued development this number will increase. We have added a paragraph to the introduction to cover these points.Methodology; why Android based smart phone app, why not iOS?An iOS app has been developed independently and will be published separately as the technology is quite different.Is data behind the app (collection of databases) freely accessible to the users, so they can download, and even verify the results and with other referenced databases? If not, then mention it in the paper, and give reasons for that. As this is an open source work, and data is collected from multiple open sources, its expected to have access to the data linked at the backend.Search results from within the app can be shared as described under “Search” in the Implementation section. Further export options are available if the full InterMine database is accessed via the app.We have added a sentence to under “search” of the Implementation section to make this clearer to the reader.Authors have mentioned list of databases, it’s important to mention licensing information of those database, to avoid any conflict of interest. Moreover, its important to clearly mention it in the conflict of interest section.All the InterMine databases are open source and provide open access to the data within them. However there are some restrictions for commercial use of some of the data and each individual InterMine should be consulted for its policy. We have added a sentence to the methods section to make this clearer to the reader.Author’s contribution are missing, it’s also important to list those.Author contributions were provided in the original paper and are available under the author details.I would suggest to write supplementary material and there explain the app in detail (step-by-step). Guide a new user as to how to get access to the app and how each interface can be used, and what are expected inputs/outputs. Furthermore, if word count restriction does not allow, then further extend supplementary material and provide comprehensive details of software implementation, database design and data workflow with rational for choosing those options. Make some diagrams (based on software engineering concepts) explaining design and implementation parts.We feel that the three key aspects of the interface have been fully explained through the use-case and accompanying screenshots. Further details for all aspects of the InterMine interface can be found in previous publications. The InterMine database design and the webservices used to power the InterMine app have also been previously described. The following sentences have been added to the manuscript to direct readers more readily to this information:Introduction (to use-case)The following use case introduces each of the three key features of the InterMine app; Gene search, Templates and Lists with an example of their use. Further details of these InterMine features have been fully described in previous publications from several InterMine databases (1,2,3,5,6,8,9).Added as first sentence under Communication:The InterMine database design and the webservices used to power the InterMine app have been previously described (1, 14)." } ] } ]	1	https://f1000research.com/articles/7-1837
https://f1000research.com/articles/8-819/v1	07 Jun 19	{ "type": "Software Tool Article", "title": "Detection and mitigation of spurious antisense expression with RoSA", "authors": [ "Kira Mourão", "Nicholas J. Schurch", "Radek Lucoszek", "Kimon Froussios", "Katarzyna MacKinnon", "Céline Duc", "Gordon Simpson", "Geoffrey J. Barton", "Kira Mourão", "Nicholas J. Schurch", "Radek Lucoszek", "Kimon Froussios", "Katarzyna MacKinnon", "Céline Duc" ], "abstract": "Antisense transcription is known to have a range of impacts on sense gene expression, including (but not limited to) impeding transcription initiation, disrupting post-transcriptional processes, and enhancing, slowing, or even preventing transcription of the sense gene. Strand-specific RNA-Seq protocols preserve the strand information of the original RNA in the data, and so can be used to identify where antisense transcription may be implicated in regulating gene expression. However, our analysis of 199 strand-specific RNA-Seq experiments reveals that spurious antisense reads are often present in these datasets at levels greater than 1% of sense gene expression levels. Furthermore, these levels can vary substantially even between replicates in the same experiment, potentially disrupting any downstream analysis, if the incorrectly assigned antisense counts dominate the set of genes with high antisense transcription levels. Currently, no tools exist to detect or correct for this spurious antisense signal. Our tool, RoSA (Removal of Spurious Antisense), detects the presence of high levels of spurious antisense read alignments in strand-specific RNA-Seq datasets. It uses incorrectly spliced reads on the antisense strand and/or ERCC spikeins (if present in the data) to calculate both global and gene-specific antisense correction factors. We demonstrate the utility of our tool to filter out spurious antisense transcript counts in an Arabidopsis thaliana RNA-Seq experiment. Availability: RoSA is open source software available under the GPL licence via the Barton Group GitHub page https://github.com/bartongroup.", "keywords": [ "RNA-seq", "antisense expression", "gene expression", "Arabidopsis thaliana", "ENCODE" ], "content": "1. Introduction\n\nAntisense RNAs are transcribed from the strand opposite to that of the sense transcript of either protein-coding or non- protein-coding genes. They appear to be widespread in all kingdoms of life and can play distinct roles in regulating gene expression or function. Typically, antisense RNAs are non-coding and expressed at lower levels than sense gene transcripts. However, they can exhibit a range of sizes, and may or may not have 5’ cap or 3’ poly(A) tails depending on whether they arise from either their own promoters, from divergent promoters, or from copying of sense transcripts by RNA-dependent RNA polymerases (see 1 and references therein,2–4). In Arabidopsis thaliana, for example, the transcription of the Flowering Locus C (FLC) gene is known to be affected by transcription of antisense ncRNAs: COOLAIR5,6, a set of ncRNAs antisense to FLC, and COLDAIR7, antisense to COOLAIR. Both COLDAIR and COOLAIR are associated with different changes in sense strand gene expression at the FLC locus8. Antisense transcription is known to affect sense gene expression through multiple mechanisms1. During transcription, RNA polymerases may physically interfere with each other if both sense and antisense transcription take place simultaneously. Interference can prevent or slow down transcription (e.g. through RNA polymerase collisions9,10) or force particular isoforms to be produced preferentially11. Post-transcriptionally, antisense transcripts can compete with sense transcripts for binding sites12. For example, the transcription of the human haemoglobin gene HBA1 is affected when the LUC7L gene on the opposite strand does not terminate, due to a deletion. It produces an antisense transcript that overlaps with HBA1, and which methylates the HBA1 promoter, repressing its expression13. In addition, since regions of protein coding genes on opposite DNA strands can overlap, their expression effectively generates transcripts that are, to varying extents, antisense to each other. Such overlapping gene pairs are a common feature of genome organization. We and others have shown that in some eukaryotic genomes tail-tail overlap enables the use of pre-mRNA 3’ processing signals in different registers for genes coded on either strand14.\n\nIncorporating antisense RNAs into genome annotation and properly quantifying their expression patterns is thus crucial, but remains challenging. Transcriptome-wide identification of RNAs is currently dominated by RNA-Seq. In this widespread experimental approach RNA is rarely sequenced directly, but instead is fragmented and first copied into cDNA and then copied again, so that libraries of DNA are sequenced. However, the copying of RNA by viral-derived reverse transcriptases is problematic. First, these polymerases exhibit DNA dependent polymerase activity, which can result in copies of the cDNA that can be incorrectly interpreted as antisense transcription. Second, just as reverse transcriptases switch template strand in viral biology, they can similarly switch templates in RNA-Seq library preparation, resulting again, in the interpretation of non-authentic antisense RNAs15–21. Historically, in microarray and RT-PCR experiments, this step is known to assign some transcripts to the wrong strand, creating spurious antisense transcripts. Preparing samples with actinomycin D can help to reduce the number of spurious antisense transcripts17 but can have unwanted side effects20. Alternative approaches to make strand-specific RNA-Seq libraries have been developed to mitigate artefacts arising from reverse transcription, however most of these also use reverse transcription22 and so have similar problems with incorrect assignment. For example, the highly-rated22,23 and widely used dUTP protocol for stranded RNA-Seq24 is known to generate low levels of spurious antisense reads ranging from 0.6–3% of the sense signal22,25,26. Ultimately, the direct sequencing of full-length RNA molecules27 will overcome many of the problems of distinguishing authentic antisense RNAs. However, currently, reverse-transcriptase based approaches dominate and the extent of spurious antisense RNAs identified in RNA-Seq datasets is rarely exposed.\n\nIn this paper, we analyse spurious antisense reads in 199 RNA-Seq experiments, across multiple organisms from both ENCODE28 and our own work. Our results show that spurious antisense reads are often present in experiments, and can manifest at levels greater than 1% of sense transcript levels. Furthermore, the number of spurious antisense reads can vary substantially between replicates within the same experiment. In some cases, this variation may be sufficient to disrupt downstream analysis of antisense gene expression, by causing spurious antisense counts to dominate the set of genes with high antisense transcription levels.\n\nTo detect and correct for wrongly assigned reads we developed a tool, RoSA (Removal of Spurious Antisense), which calculates an antisense correction factor by identifying subsets of reads where all antisense reads are spurious. We evaluate the effect of using RoSA on Arabidopsis thaliana experimental data where varying levels of spurious antisense were present in different replicates. RoSA reduces the overall dependence of antisense counts on sense counts, a key indicator of the presence of spurious antisense. For individual genes with different real and spurious antisense characteristics, RoSA reduces spurious antisense counts while retaining the antisense signal.\n\n\n2. Methods\n\nAs noted by Jiang et al. (2011,25), spurious antisense read counts can be estimated by analysing either ERCC spike-in data or counts of sense and antisense reads around splice sites. Each approach has different advantages: using spike-ins is simpler and faster, while using spliced reads allows a gene-by-gene estimate to be made. RoSA implements both approaches, in conjunction with pre-processing scripts to generate specialised read counts required by the tool. Once RoSA has an estimate of the levels of spurious antisense, it can adjust the raw antisense counts to account for the incorrectly stranded reads.\n\nOur scripts and analysis code are bundled as a tool, RoSA (Removal of Spurious Antisense), available from the Barton Group’s github pages at https://github.com/bartongroup/RoSA. RoSA is an R package supported by two python pre-processing scripts, callable from R.\n\nFor genes with spliced transcripts which are expressed in the data, RoSA uses the subset of reads from either strand that map across the splice junctions. The antisense reads in this subset are almost certainly spurious, and so RoSA can use the read counts to calculate a gene-specific antisense correction factor (Section 2.2). For genes without spliced transcripts, RoSA uses ERCC spike-in data, if present. Here any antisense read mappings are, by definition, spurious and the ratio of sense to antisense reads mapping to the spike-ins thus provides a global, rather than gene-specific, antisense correction factor (Section 2.3). If ERCC spike-in data is not available, RoSA instead calculates a global estimate of the spurious antisense fraction from the set of spliced reads. Counting all, or spliced-only, antisense reads is not directly supported by existing tools. RoSA’s pre-processing scripts perform these functions. The make_annotation script creates an antisense annotation (as gtf) from a standard annotation (as gff or gtf), which can then be used to generate antisense read counts via a standard read counting tool (Section 2.4.1). RoSA doesn't specify how the sense and antisense gene expression is counted leaving users free to apply whichever tool they feel will best represent the gene expression in their experiment. However, the accuracy of the corrections calculated by RoSA will be affected by this choice in the same way as the calculation of differential gene expression. If counting methods are used that only consider regions within sense features that do not overlap any antisense feature, the gene-specific corrections calculated by RoSA may be less accurate where the overlap is large and/or the sense or antisense expression is low.\n\nRoSA then adjusts these raw counts to produce corrected antisense counts (Section 2.4). The count_spliced script generates sense and antisense counts of reads at splice junctions, used when estimating spurious antisense from spliced reads. The script takes a standard annotation (as gtf/gff) and corresponding alignment (as bam) and outputs counts of spliced sense and antisense reads to a designated output file.\n\nRoSA takes several datasets containing different read counts as its input, for each replicate:\n\n1. Full read counts by gene\n\n2. Antisense counts by gene (via the make_annotation script)\n\n3. At least one of:\n\na. Spike-in sense and antisense counts\n\nb. Spliced sense and antisense counts (via RoSA's count_spliced script)\n\nRoSA calculates and returns antisense:sense ratios for the spike-in data, or spliced read data, or both, and, for each gene and replicate, outputs new read counts values corrected for spurious antisense. RoSA also plots antisense versus sense counts of the original and corrected data, by replicate.\n\nRoSA’s main approach to estimating spurious antisense is to use spliced reads within the main dataset. Reads which map antisense to a multi-exon gene, and that also show the same splicing pattern as spliced sense-mapping reads are almost certainly spurious, as the splicing motif (canonically GU-AG) will be incorrect on the opposite strand (Figure 1). An estimate of spurious antisense can be calculated by considering only spliced reads whose splices match annotated splice sites (splice-matched reads), and, as with the spike-ins, calculating the ratio of antisense to sense reads.\n\nThe reverse strand gene AT4G18970 is strongly expressed in all 3 replicates (bottom tracks). Spurious antisense can also be seen in all replicates (top tracks), with splice points in the antisense signal matching splice points in the sense signal. Furthermore, the level of spurious antisense varies noticeably between replicates. (Figure generated by IGB32).\n\nSplice-matched reads are identified by first filtering all spliced reads in the data. In a bam file of aligned reads, spliced reads have a CIGAR string containing ‘N’, indicating a skipped region. SAM processing tools such as sambamba29 support filtering on the CIGAR string and can extract spliced reads rapidly. A second filtering step pulls out only those reads whose splice locations match at least one intron in the annotation, by processing each read in turn, identifying the spliced positions (based on the read location and the CIGAR string) and checking the annotation for a matching intron. Finally, the strand of each spliced read can be determined from its flag field value30, and compared to the strand of the matching intron(s). Reads on the same strand as the intron(s) are counted as sense reads, and remaining reads as antisense reads. Since spurious antisense reads are misallocated sense reads, the number of antisense splice-matched reads assigned to a gene is strongly positively correlated with the number of its sense splice-matched reads (see Section 3). The ratio of antisense:sense counts on the splice-matched reads thus gives a simple global estimate of the level of spurious antisense across the whole dataset. Using spliced reads has the advantage that an antisense:sense ratio can be calculated on a gene-by-gene basis, for any spliced gene. Genes without any spliced reads fall back on the global estimate, calculated either from the spike-ins (see Section 2.3) or the spliced reads.\n\nIn the case of real, unannotated, antisense expression at a gene locus, the behaviour of RoSA falls into three categories:\n\n1. If the splicing of the true antisense transcript differs from the sense transcript (including no splicing) then RoSAs gene specific correction will remove any spurious antisense expression (identified by antisense matches to the sense splicing) and leave the true antisense expression unchanged.\n\n2. If the splicing of the antisense expression is the same as the sense strand, then RoSA will remove this completely.\n\n3. If the true antisense splicing is the same as the sense strand in some parts of the transcript, but not across the entire transcript, then RoSA will remove a fraction of the true antisense expression depending on how similar the splicing patters are.\n\nWe anticipate that occurrences of 2 & 3 will be uncommon in RNA-seq datasets. Point 2 highlights a minor potential limitation of the gene-specific splicing-based corrections calculated by RoSA, namely that it cannot distinguish between spurious anti-sense signal and potential biological transcription from RNA-dependent RNA polymerases (RdRPs). Although RdRPs are widespread in eukaryotic genomes, only 8–30% of eukaryotic gene regions have significant length ORFs on their opposite strands31, providing an upper limit on the potential impact of this method of transcription on the RNA complement within a cell. Eukaryotic RdRPs evolved independently from their viral counterparts and, in plants, are involved in siRNA transcriptional silencing33. This is not the case in animals however (except in C. elegans) where their function remains elusive34.\n\nAn alternative approach to estimating spurious antisense is to use ERCC spike-in data. Developed by the External RNA Control Consortium (ERCC)35, the ERCC spike-in controls are synthetic RNA transcripts that are added to RNA-Seq experiments to act as controls36. The 92 spike-ins are designed to mimic a range of eukaryotic mRNA characteristics, varying in length, GC-content and concentration, with a 20bp poly-A tail. They have minimal sequence similarity with known eukaryotic transcripts. Since the spike-ins are synthetic, they are unidirectional, and so any reads assigned as antisense to a spike-in can be assumed to be spurious. As the spike-ins are present at a wide range of concentrations, they are detected with a wide range of read counts, permitting an estimate of the ratio of antisense to sense read counts on the spike-ins to be calculated, which can then be used to estimate the contribution of spurious antisense transcripts across the full dataset. Obtaining sense and antisense counts for the spike-ins is straightforward. First we align the reads to the spike-ins (using the spike-in annotation file ERCC92.gtf, available at https://www.thermofisher.com/order/catalog/product/4456739) and then count reads, using a strand-aware read counting tool such as featureCounts37, HT-SeqCount38, etc. Now averaging the spurious antisense:sense ratio across all of the spike-ins gives a global estimate of the spurious antisense, in just the same way as for the spliced reads.\n\nHaving identified high or differing levels of spurious antisense in an RNA-Seq experiment, we also want to correct for the incorrectly assigned reads so that true differential expression calling can be performed. The ratio of spurious antisense:sense read counts can be used as a simple correction factor. Defining r as the ratio of spurious antisense:sense and S and A respectively as the number of sense and antisense counts for a gene, the number of spurious antisense read counts AS is estimated for each gene as: AS = r.S .\n\nThen the antisense count can be corrected to account for the spurious antisense by taking A - AS. This correction simply adjusts read counts for each gene, and does not identify specific reads as incorrectly assigned, so pile-ups cannot be adjusted. Since the spurious antisense reads are mis-assigned sense reads, RoSA then adds the spurious antisense count for each gene to its sense count.\n\n2.4.1 Counting antisense reads. In order to apply the antisense correction factor, counts of antisense reads for each gene are required. Counting antisense reads is not directly supported by read counting tools. However, it can be performed with featureCounts37 by setting parameters to indicate that reads are stranded in the opposite direction to which they are. Unfortunately, if there are overlapping genes then reads in the overlaps will be counted twice using this tactic. As reads in regions of gene overlap are necessarily ambiguous, they cannot be considered to be antisense, spurious or otherwise. RoSA avoids this issue by building a custom antisense annotation based on the input sense annotation but excluding regions where genes on opposite strands overlap. Different gene transcripts are accounted for by merging all transcripts for a gene into a single maximal transcript. Whenever exons of different transcripts overlap in the annotation, the exon in the maximal transcript is the maximum extent of both exons. Given a maximal transcript, the script creates an antisense feature on the opposite strand which runs for the full extent of the maximal transcript. If the maximal transcript of another gene overlaps with the antisense feature, then the antisense feature is truncated to avoid overlapping. Once an antisense annotation is available, a read counting tool can be used to count antisense reads, by providing the antisense annotation instead of the standard annotation.\n\nA procedure to experimentally generate RNA-Seq data with specific levels of spurious antisense is not known. Our main experimental data (Experiment 1) is therefore drawn from the study which originally motivated our investigation into incorrectly assigned antisense reads. In this study, spurious antisense occurred by chance at varying orders of magnitude across different replicates. Additionally, we perform a meta-analysis using three other Arabidopsis thaliana datasets (Experiments 2–4,39) and data from ENCODE (see Underlying data for the full list of the ENA and ENCODE accessions).\n\n2.5.1 Arabidopsis sample preparation and sequencing. Briefly, the RNA-Seq data for Experiments 1 is wild-type (WT) Arabidopsis thaliana Colombia-0 (Col-0) biological replicates. WT A. thaliana Col-0 seeds were sown aseptically on MS10 plates. The seeds were stratified for 2 days at 4°C and then grown at a constant 21°C under a 16-h light/8-h dark cycle for a further 14 days, at the end of which the seedlings were harvested. Total RNA was isolated from the seedlings with the RNeasy Plant Mini Kit (Qiagen). In Experiment 1, DNAse digestion was performed on column, as a part of RNA isolation, and 8 μl of ERCC spike-ins (External RNA Controls Consortium 2005) at a 1:100 dilution was added to 4 μg of total RNA. Libraries were prepared according to the TruSeq® Stranded mRNA Sample Preparation Guide Rev E. The libraries were sequenced on a HiSeq2000 at the Genomic Sequencing Unit of the University of Dundee. This preparation largely mirror the sample preparation of the datasets take from Froussios et al. (2017,39, Experiments 2–4) In Experiments 2–4, however, the sequencing libraries were prepared using the Illumina TruSeq® Stranded Total RNA with Ribo-Zero Plant kit.\n\nExperiment 1 has 3 replicates, processed as one batch, with a total of 4 × 108 150-bp paired-end reads. Experiments 2 and 3 have 7 biological WT replicates, while Experiment 4 has 3, for a total of 17 biological WT replicates and ~1.7 × 109 100-bp paired-end reads across the three experiments. The same lab sowed, grew and harvested the plants, and prepared the libraries. The sequencing was performed on the same machine by the same people at the same sequencing facility and all the samples include the ERCC spike-ins which can verify the WT samples are consistent and comparable across experiments.\n\n2.5.2 Quality control, alignment and quantification. The quality of the data was quantified using FastQC v0.11.240 with all the replicates performing as expected for high quality RNA-Seq data with good median per-base quality (≥28) across >90% of the read length. The read data for all experiments were aligned to the TAIR1041 Arabidopsis thaliana genome using the splice-aware aligner STAR v2.4.2a42 for Experiment 1 and STAR v2.5.0 for Experiments 2–4. The index was built with --sjdbOverhang 149 (Experiment 1) or –-sjdbOverhang 99 (Experiments 2–4) and the alignment was run with parameters: --outSJfilterIntronMaxVsReadN 5000 10000 15000 --outSAMAttributes All --outFilterMultimapNmax 2 --outFilterMismatchNmax 5 --outFilterType BySJout.\n\nThe read data were also aligned to the ERCC spike-ins annotation, using the same parameters. Read counts per gene were then quantified from these alignments with featureCounts v1.5.0-p1 using the publicly available TAIR10 annotation with the parameters: -s 2 -p -t exon --largestOverlap. After running RoSA’s make_annotation script to build an antisense annotation, antisense read counts per gene were quantified in the same way, with the parameters: -s 2 -p -t antisense --largestOverlap. Finally, spliced sense and antisense reads were counted using RoSA’s count_spliced script with the TAIR10 annotation.\n\nA full description of RoSAs environment, dependencies, installation and basic operation can be found on the RoSA GitHub repository. Briefly, RoSA is a combination of an R package and python scripts for data preprocessing. Minimal system requirements for the package are R v3.5+, python 2 v2.7+ the LSD R package and the python packages scipy (v0.16.1 - 0.17.1), numpy, pandas (not v0.20.1), six and, optionally, drmaa for cluster integration. The python scripts to find and count spliced antisense and sense reads also depends on sambamba. To facilitate ease-of-use, a conda environment that captures all the relevant dependencies is included as part of the RoSA codebase. RoSA’s python scripts are provided as a python package ad are installed via pip, while the R package can be installed directly from within R using the devtools package.\n\nRoSA operates on the total and spliced read counts from sense and antisense bam format read alignments of stranded RNA-Seq datasets, either with or without ERCC spike-in standards. To facilitate easy generation of this read count data, RoSA includes helper pre-processing scripts to generate the antisense counterpart of the provided gtf/gff format sense-strand genome annotations (make_annotation), and to generate spliced-read gene count data from the bam format read alignments using both the sense- and anti-sense annotations (count_spliced). Both of these helper scripts can be called directly within R as part of the RoSA R package, Detailed help for the R RoSA functionality can be accessed within R with the command, help(rosa).\n\n\n3 Results\n\nWe used RoSA to analyze our data from Experiment 1 for spurious antisense, using both the spike-in and spliced reads counts. RoSA calculated antisense:sense ratios for the spike-ins (Figure 2) showing that the 3 replicates have antisense:sense ratios on the spike-ins of 0.0008, 0.004 and 0.011. Although these ratios are small, if the replicates were being compared for differential expression, the differences are potentially substantial for highly expressed genes, and could lead to differential antisense expression being called erroneously.\n\nPoints represent antisense and sense read counts for individual spike-ins. Each line is the average antisense:sense ratio for one replicate. Here, antisense:sense ratios vary by an order of magnitude across the 3 replicates, with values of 0.004 (WT1), 0.0008 (WT2), and 0.011 (WT3).\n\nFor each replicate we calculated the spurious antisense:sense ratios for the spliced reads with RoSA, and compared them to the spike-ins. An overview of the results for all three replicates shows that the spurious antisense levels calculated from the spike-ins are in good agreement with the levels calculated from the spliced reads (Figure 3 and Figure 4, Row 1).\n\nRatios estimated from spike-ins show good agreement with ratios estimated from spliced reads. Outliers not shown.\n\nEach column presents data for one replicate. Row 1: Antisense:sense ratios calculated from spike-ins (Black points & fit line) and spliced reads for each gene (density heatmap). The antisense:sense ratios for both the spike-ins and spliced reads are in good agreement. The strong correlation between the sense and antisense spliced counts, and the constant antisense:sense ratio across all genes, indicates that the majority of the antisense expression in the data is not a sequence-, or gene-specific, phenomenon. Rather, this is what would be expected from a systematic process affecting a constant fraction of the sequenced reads. Row 2: Antisense:sense ratios calculated for the full gene counts (spliced & unspliced). The correlation between the sense and antisense expression persists, however it is weaker than the correlation using just the spliced and spike-in sense and antisense expression. This reflects the inclusion of true biological antisense expression, unspliced genes where a global correction is less accurate, and low expression genes where the splicing correction is not well measured. Row 3: Corrected antisense:sense ratios calculated for the full gene counts (spliced & unspliced). The corrected antisense counts show much weaker correlation with the corresponding gene counts reflecting the removal of the systematic spurious antisense count signal. On all plots the dashed line marks y=x; points above this line correspond to genes where the antisense:sense ratio is > 1.\n\nFinally, RoSA calculated a spurious antisense correction across the whole of each replicate. Every spliced gene was corrected with the antisense:sense ratio specific to the gene, and unspliced genes were corrected using the mean ratio calculated from the spike-ins. (RoSA also allows the unspliced correction to be calculated from the mean spliced reads ratio, for datasets without ERCC spike-ins). Overall, RoSA reduces the correlation between antisense and sense counts in the data (Figure 4, Rows 2 & 3), as would be expected with a reduction in incorrectly assigned reads. Two examples of corrections made by RoSA are shown in Figure 5, where the antisense signal appears to be almost entirely spurious, RoSA’s correction factor reduces the antisense counts substantially, but where there also appears to be some real antisense signal, RoSA’s correction factor leaves a higher proportion of counts.\n\nThe reverse strand gene AT4G18970 (left) has antisense expression which clearly matches the splice sites of the sense strand. RoSA eliminates almost all of the antisense reads. The forward strand gene AT5G66570 (right) has both antisense expression matching the sense strand splice sites, and a peak at the 5’ end which is unlikely to have resulted from incorrect read assignment. RoSA only reduces the antisense counts by around 40%. (Figures generated by IGB32).\n\nAs well as identifying instances of antisense expression, looking at antisense counts in this way can also be useful in identifying misannotated genes. For example, in our data there are many genes where the antisense:sense ratio is more than 1 (e.g. see points lying above x=y in Figure 4, Row 2), which may indicate an incorrect strand assignment in the annotation.\n\nCalculating antisense:sense ratios allows comparisons of spurious antisense to be made between replicates and between experimental condition, and can reveal whether there are systematic differences which might confound experimental comparisons. For example, Figure 1 presents results from an RNA-Seq experiment where spurious antisense levels differed by an order of magnitude between replicates. In this experiment, the WT replicates had spurious antisense:sense ratios of 0.0031 (SD 0.0116), 0.0009 (SD 0.0070) and 0.0111 (SD 0.031).\n\nTo determine the extent of this problem for RNA-Seq datasets in general, we investigated the spurious antisense levels across a range of experiments and research groups. We analysed antisense reads assigned to the spike-ins from three other experiments in our lab (Experiments 2–4), as well as 195 publicly available human datasets from the ENCODE project that included the ERCC spike-ins28 (see Underlying data for details of the sense, antisense and RoSA-corrected antisense expression for all A. thaliana genes in the datasets from Experiments 1–4). A separate antisense:sense ratio was calculated for each replicate in each experiment (Figure 6), showing that spurious antisense reads are present at varying levels and can range across several orders of magnitude. This presents a serious quality control issue for anyone investigating differential antisense expression: a real difference in antisense expression could be completely masked by a difference in spurious antisense.\n\nData are from either from ENCODE (Gingeras, Graveley and Lecuyer) or our own group (Simpson). Each point represents the ratio for a replicate. Ratios range from 0.0111 to 0.00003.\n\n\n4 Conclusions\n\nSpurious antisense is common in strand-specific RNA-Seq datasets and can occur at varying levels across replicates in the same experiment. Differing levels of such incorrectly assigned reads are enough to disrupt differential expression analyses of antisense gene expression.\n\nWe have developed a new tool, RoSA, which can detect, quantify and correct for spurious antisense. RoSA provides an important quality control step for researchers analyzing antisense expression in their data.\n\n\nData availability\n\nArabidopsis col-0 WT strand-specific RNA-Seq data from poly-A pulldown, Accession number E-MTAB-7990: https://identifiers.org/arrayexpress/E-MTAB-7990\n\nRNA-seq data of wild type Arabidopsis seedlings, Accession number E-MTAB-5446: https://identifiers.org/arrayexpress/E-MTAB-5446\n\nZenodo: bartongroup/RoSA: Initial, http://doi.org/10.5281/zenodo.266137843.\n\nThis project contains the following extended data:\n\n- Accession numbers for ENCODE data: https://github.com/bartongroup/RoSA/tree/master/extras/F1000_manuscript/RoSA_Extended_Data.docx\n\n- Accession details for ENCODE data: https://github.com/bartongroup/RoSA/blob/master/extras/F1000_manuscript/ENCODE_accessions.xlsx\n\n- Arabidopsis seedlings RNA-seq read count expression counts: https://github.com/bartongroup/RoSA/tree/master/extras/F1000_manuscript/expression_data.csv\n\nLicense: GNU General Public License 3.0.\n\n\nSoftware availability\n\nSource code available from: https://github.com/bartongroup/RoSA\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.266137843.\n\nLicense: GNU General Public License 3.0.", "appendix": "Grant information\n\nThis work has been supported by the Biotechnology and Biological Sciences Research Council [BB/M004155/1, BB/M010066/1] to G.J.B. and G.G.S.\n\nAll funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nA previous version of this article is available on BioRxiv: https://doi.org/10.1101/425900.\n\n\nReferences\n\nPelechano V, Steinmetz LM: Gene regulation by antisense transcription. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFroussios K, Schurch NJ, Mackinnon K, et al.: How well do RNA-Seq differential gene expression tools perform in a eukaryote with a complex transcriptome? bioRxiv. 2017. Publisher Full Text\n\nAndrews S: FastQC: A quality control tool for high throughput sequence data. 2010. Reference Source\n\nArabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000; 408(6814): 796–815. PubMed Abstract \| Publisher Full Text\n\nDobin A, Davis CA, Schlesinger F, et al.: STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1): 15–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchurch N: bartongroup/RoSA: Initial (Version v1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2661378" }	[ { "id": "49670", "date": "02 Jul 2019", "name": "Ian Sudbery", "expertise": [ "Reviewer Expertise I am a lecturer in bioinformatics. My research focuses on using the tools for computational biology and functional genomics to study RNA biology and regulation of gene expression", "particularly at the post transcriptional level." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMourao et al. describe a package for estimation of the rate at which stranding information in an RNA-seq sample might be wrong, thus leading to inaccurate quantification of anti-sense transcription, which they apply to their own data and the data of others. This is a fascinating and useful contribution to RNA-seq analysis. The software described should be useful to a range of scientists working the analysis of RNA biology from transcriptomics data. I will split my evaluation of this into two sections – the manuscript/science, and the software:\n\nManuscript:\nThe manuscript is generally well written and describes an interesting problem. I believe that the approach is generally sound. I have a few small issues that the authors might like to address:\nIt is assumed that anti-sense spliced reads are spurious. However, one possibility is that anti-sense spliced reads arise by the action of RNA-dependent RNA-polymerases. The authors acknowledge this point, but state “only 8-30% of eukaryotic gene regions have significant length ORFs on their opposite strands, providing an upper limit on the potential impact of this method of transcript on the RNA complement within a cell”. I am unsure how the presence of an ORF on the anti-sense strand has an effect on the RNA complement of a cell. The authors should clarify this argument.\n\nAn underlying assumption of the approach is that the ratio of spurious anti-sense reads to sense reads is consistent across the length of the transcript, and thus modelled by the reverse junction reads. This assumption is probably necessary and reasonable, but perhaps the authors might make it explicit.\n\nIn regions where two annotated transcripts overlap, the authors truncate their antisense copies of these transcripts to only cover the region that is unique to the sense transcript in question. This is necessary to avoid double counting of reads. However, it does mean that the number of reads counted as antisense will be an underestimate. This becomes problematic because the full length transcript, not the truncated version is used when calculating how many spurious antisense reads are present (this is possible because this is calculated from spliced reads). Thus the correction factor will be overestimated compared to the counted number of antisense reads. This could be detected by finding genes where the correction factor is larger than the total number of antisense reads leading to a negative corrected value for the antisense count. Unfortunately the package truncates the corrected count at 0, so these counts are not evident in output from the package.\n\nThe authors show that there is a large variation in the proportion of anti-sense reads that are spurious in three replicates of the same experimental condition. It would be useful to know if this variation is bigger or smaller than that between conditions. Variation within replicates reduces power to detect differential expression between conditions, but systematic variation between conditions might lead to false positive calls of differential expression. The authors might consider looking at the within condition and between condition variance of samples in the Graveley data set they have analysed.\n\nThe authors introduce the concept of a “maximal transcript”. This is very similar to the concept of the superTranscript1 the authors might like to acknowledge this and cite the relevant work.\n\nThe authors note that for some genes there is more anti-sense expression than sense expression and suggest this might be a case of incorrect annotation. Do these genes tend to be spliced or single exon. If spliced, does the annotation carry the canonical splice sequence, its reverse complement, or some other sequence?\n\nFigure 5 is reproduced at a very low resolution, such that it is not possible to read all the text on the panel marked (Right).\nSoftware:\nThe software is provided as an R package with a number of accompanying python modules and is available on GitHub. An archived version of the source code used to produce the figures in the paper is available on Zenodo. Dependencies are intended to be installed using a conda environment. The authors are to be commended on their good practice in this respect.\nHowever, I did have a number of issues in attempting to use the software:\nI could not install the conda environment from the provided environment file. Running the suggested commands gave the following error:\nSolving environment: failed ResolvePackageNotFound:\n\n- r-base==3.5.1=h4fe35fd_0\n\n- libssh2==1.8.0=h5b517e9_2\n\n- matplotlib==2.2.3=py27h8e2386c_0\nTo try and fix this problems I removed the pinning on the exact build, leaving only the pinning on version number. This returned the following error: Solving environment: failed\nUnsatisfiableError: The following specifications were found to be in conflict:\n\n- matplotlib=2.2.3\n\n- sip=4.18.1\n\n- tk=8.6.8\nI was able to install by manually creating an environment using the dependencies listed in the readme, although using the pip install for rosa downgraded the pandas install from 0.24 to 0.19.\nThe ability to install all dependencies as a conda environment is very useful to the average user, and the authors should consider either fixing the supplied env file, or (ideally) creating a proper rosa conda package, so that rosa and all its dependencies (both R and python) can be installed with conda install rosa.\n\nOnce installed I found the documentation to be a little on the sparse side, and it times confusing. For example, the help for the `rosa` function states that the parameter “data” should be a list of dataframes with a particular list of columns. But the help for the “group” parameter says that it is a character vector specifying the replicates for each column in data. Thus it is unclear if replicates should be provided as a member of a list of data.frames, or as separate columns in one data.frame.\n\nThere are required arguments for matrices of spike-in counts, as well as an argument to provide the positions of spike-ins in the “data” matrix. It is unclear whether spike-ins should be provided using only one or both of these mechanisms, or what to do if there is no spike-in data.\n\nThe help states “This function was written with the intention of obtaining count and group parameters from an edgeR DGEList object d, via d$counts and d$samples$group.” But d$counts would not return a data.frame of the format specified in the help for the “data” argument.\n\nThe manuscript doesn’t really mention TPMs, but the help for rosa states that TPMs are required to run the function. I believe it would not be clear to a less experienced user how to calculate a TPM for the anti-sense counts generated on the custom anti-sense annotation.\n\nNot help page is provided for either the `count_spliced` nor `make_annotation` functions. Nor is much in the way of documentation provided for the python scripts these functions call.\n\nThe package has no associated vignette. I think it would be of great use to users, particularly less experienced ones, to provide a walk-through of the analysis of a dataset from start to finish, with a simplified example dataset.\nOverall, I think that this work is both novel, interesting, useful and basically sound. Before I can recommend approval, I would need to see minimally that the issues identified with installation and documentation have been dealt with. I would also be interested in the authors replies to my comments on the manuscript itself.\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": "52108", "date": "28 Aug 2019", "name": "John Casement", "expertise": [ "Reviewer Expertise Andreas Werner: Molecular biology", "natural antisense transcripts. John Casement: Bioinformatics", "RNAseq 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 address an important question, i.e. detection of real and experimentally generated antisense transcripts in strand-specific RNAseq datasets. With their software tool RoSA, they present a valid strategy to solve this problem. However, there are a number of concerns regarding the manuscript and the software.\n\nManuscript:\nIntroduction: The authors discuss the mechanisms of how antisense transcripts can regulate/interfere with the expression of the sense gene. The sentence ‘Post-transcriptionally, antisense transcripts can compete with sense transcripts for binding sites’, may be applicable in bacteria, but is misleading when studying eukaryotic systems. Post-transcriptional mechanisms involve RNAi, RNA editing or RNA-protein complexes but hardly competition for binding sites with the sense transcript. Moreover, the example given in the text (research by Tufarelli et al.1) is a prime example of transcriptional interference, not of post-transcriptional regulation.\n\nIntroduction: ‘viral-dependent‘ should read ‘virus-dependent’.\n\n2.2 Using spliced reads: The fact that RoSA cannot distinguish between spurious antisense reads and RdRP generated transcripts are only relevant if datasets from plants and C.elegans are interrogated (that express significant levels of RdRP). The issue becomes critical if particular loci affected by RdRP amplification contribute disproportionately to a genome wide correction factor. In line with reviewer 1, it is unclear how an open reading frame affects RNA dependent RNA polymerization.\n\n2.4 Mitigating spurious antisense: ‘Since the spurious antisense reads are miss-assigned sense reads, RoSA then adds the spurious antisense count for each gene to its sense count’. Adding the spurious antisense counts to the sense counts assumes that reverse transcriptase errors happen at the cost of ‘real’ RNA -> cDNA processing. Is this correct?\n\nSoftware:\nThe package as it stands is not user friendly and has several bugs that need to be resolved before the software can be used smoothly.\n\nInstallation of the Python package using the pip installer worked without difficulties, as did the R package installation.\n\nRequesting help at the command-line (via the -h option) for the Python scripts 'make_annotation' and 'count_spliced' had the side-effect of creating an empty log file each time.\n\nRunning the 'count_spliced' script over a modestly sized alignment file (around 0.5 GB) resulted in over 2000 log files being produced, each detailing the locations at which spliced reads were being counted. This may be related to running the script without drmaa (in serial mode), as a message to that effect was repeatedly printed to the terminal during operation.\n\nThe Python scripts can be run as R functions, but the help documentation is not available within R.\n\nA detailed R package vignette including sample data sets and examples of code would be very helpful, especially for the less experienced users.\n\nGeneral comment: Is there a reason why the authors used a storage costly strategy aligning reads followed by counting rather than using alignment free software such as Kallisto or Salmon?\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": [] } ]	1	https://f1000research.com/articles/8-819
https://f1000research.com/articles/8-818/v1	07 Jun 19	{ "type": "Research Article", "title": "Drug use among agriculture-related workers in Thailand", "authors": [ "Narumon Janma", "Manop Kanato", "Poonrut Leyatikul", "Narumon Janma", "Poonrut Leyatikul" ], "abstract": "Objectives: To examine drug use prevalence and to explore the associations of cluster environment characteristics with drug use among agriculture-related workers in Thailand. Methods: This was a cross-sectional study involving 2936 agriculture-related workers from 10 clusters in 4 regions throughout Thailand. Trained interviewers conducted semi-structured interviews. Additionally, the drug-use patterns and behaviors of 124 current users were structurally observed. A multilevel binary logistic regression model was used to estimate the effects of the cluster environment on drug use. Results: The annual prevalence was 58.73%. Illicit drugs, non-prescription drugs, or over the counter medicines were widely used. Age, sex, and non-prescription behavior were associated with substance use that was statistically significant. Contextual clustering was found to significantly affect drug use among agriculture-related workers. A 1-unit increase in treatment rate predicted 12.7-times higher illicit drug use and 15.3-times higher methamphetamine use. Conclusions: Agricultural work facilitated the spread of drug use The design of the surveillance system should be considered.", "keywords": [ "drug use", "agriculture-related workers", "Thailand" ], "content": "Introduction\n\nIllicit drug (ID) use has been widely recognized as a challenge to both individual health and society. According to the United Nations Office on Drugs and Crime, 5.6% of adults (15–64 years) across the globe used IDs at least once in 2016 and >10% of those drug users have suffered from drug-use disorders1. In the Association of Southeast Asian Nations (ASEAN), the rate of drug users in 2016 who accessed treatment systems was 27.8 per 100,000 people. Over 80% of treated people were methamphetamine users2. A 2016 household survey in Thailand revealed that 1.4 million Thai people aged between 12–65 years used drugs, 2.8% of the adult Thai population. The results from the last Thai national survey showed that cannabis, kratom (Mitragyna speciosa Korth), and methamphetamine were the three most commonly used drugs among ID users3. Government statistics reported that only 13% of these drug users were estimated to access all treatment systems in 20164. Although drug use in Thailand has been lower than the global average, the problem has been a part of the national agenda since 20015.\n\nSince 1360, Thailand has been affected by the problem of illicit substance use6. Common indigenous plants from which IDs are sourced are opium, cannabis, and kratom. The first synthetic drug used in the population was heroin, first noticed in the early 1960s. The use of stimulants (amphetamine and methamphetamine) evolved into a major epidemic in the early 1970s. Volatile substances (benzene, lacquer, and glue) appeared in the late 1970s7. In the late 1990s, a new set of substances, including ecstasy, ketamine, crystalline methamphetamine, and cocaine, began to be widely used8. Recently, the use of non-medical pharmaceutical drugs and new psychoactive substances has become evident9. However, official statistics show that methamphetamine users were the biggest group of drug users who accessed treatment10. Since cannabis and kratom are less harmful than other drugs, only a few users have required treatment.\n\nIn general, people take drugs for a variety of reasons. Psychostimulants are thought to reduce fatigue and enhance activities11,12. Methamphetamine, which is an amphetamine-type stimulant (ATS), has a long history of use in the Asia-Pacific region13. Presently, among ASEAN member states, methamphetamine is distributed in crystal, powder, and pill forms14.\n\nIn Thailand, ATSs have been known since the early 1960s and are thought to have been a major problem since the 1990s15. Recent statistics show that three quarters of ATS users are methamphetamine users10. Additionally, two-thirds of drug users who sought treatment from the government system were laborers, including agriculture-related workers. Agricultural products accounted for 8.7% of Thailand’s annual GDP16. Of all workers employed in 2016, 30% were agriculture-related workers17. Recent studies have reported significant associations between substance use and agricultural activities18,19.\n\nStudies have found that substance abuse behavior was affected by various social determinants, including personal characteristics and relationships with peers/family and the neighborhood20–22. Some studies have focused on individual and interpersonal factors related to substance abuse21,23. Other studies have explored the extent of neighborhood factors that influence the risk of relapse21,24–26. A few studies in Thailand have reported how social factors (a combination of individual, peer, and family factors) influenced substance use27,28.\n\nAlthough evidence has emerged showing that agriculture-related workers potentially use drugs, the prevalence of drug abuse and neighborhood cluster association was unclear. There were two objectives in this study: to explore the proportion of drug users among agriculture-related workers and to investigate the association between cluster-level factors and substance use among agriculture-related workers by using model simulation following the method described by Bronfenbrenner29.\n\n\nMethods\n\nThis cross-sectional study was conducted from September 2017 to July 2018.\n\nIn 2017, a total of 5,911,567 individuals had registered as agricultural-related workers in Thailand30. Only workers who had worked for the past 3 months were eligible to participate in the survey. The prevalence of substances used from the latest national household survey in 2016 was used as a study parameter to calculate sample sizes3. The sample size calculation method for prevalence survey31 was employed with adjustment of design effect from multistage sampling. With an expected 10% non-response and ±1%% error, a sample size of 3140 individuals was targeted. Stratified multistage cluster sampling was employed. Thailand was stratified into 10 geographical zones on the basis of governmental narcotic operation areas; each zone comprised 7–12 provinces. Each zone was a systematically sampled province (one zone per province). The 10 sample provinces were Chiangmai, Phitsanulok, Khon Kaen, Nakornrachasima, Samitprakarn, Chonburi, Samutsakorn, Krabi, Satun, and Bangkok. Then, the agricultural clusters of each province were mapped. Only one cluster was randomly selected from each province, which gave 10 clusters altogether. Households were systematically selected from the updated map of individual clusters. The number of households was determined on the basis of the probability proportional to size. Simple random sampling using a table was employed to obtain a sample from each household (one household per sample). Of 3140 intended samples, 2936 (93.5%) individuals agreed to participate in the survey (ages ranging from 16 to 69 years old, with an average of 36.6 years old; 61.3% (1799) were males).\n\nAfter the baseline survey, 124 current methamphetamine users agreed to participate in structured observation regarding their drug use (age ranging from 18 to 58 years old, with an average of 27.4 years old; 88.7% (110) were males).\n\nOutcomes of the study were identified as drug use prevalence. In this study, drug use was defined as the use of illicit drugs (IDs) (heroin, cannabis, methamphetamine, etc.); legal substances, such as non-prescription drugs alcohol, tobacco and caffeine, were not included. Of all IDs, methamphetamine is hypothesized to be widely used among agricultural rated workers to accelerate productivities resulting in higher prevalence. Annual prevalence as well as past month prevalence was obtained.\n\nThe interview questionnaire and structured observation guideline were developed by 12 experts32. The interview questionnaire comprised individual-level, interpersonal-level, and cluster-level variable sections, as well as substance use. The structured observation guideline emphasized pattern of methamphetamine use and its effects.\n\nA total of 20 research assistants (two for each province, one male, one female) were trained to conduct field work (updating a cluster map, devising a household sampling frame, randomly selected samples, requesting consent, and interviewing the samples). The research assistants asked the intended participants to provide written informed consent, waited a couple of days to allow the participants to make their own decision, and, then, made an appointment for them to come back to be interviewed/observed. Data from the survey were gathered from September to November 2017 via private, face-to-face interviews. Interview guides are available as Extended data. The participants chose a convenient place and time for their interviews (e.g., the participant’s household). An average of 1–1.5 hours was spent for each interview. After the interview, the research assistants requested written consent to participate in structured observation of the drug users. Only those who consented (n=124) were observed regarding their drug-use behavior—such as when and how IDs were used, and the amount and frequency used—and its effects. The research team conducted participant observations from November 2017 to July 2018 (3–5 times for each sample, 1–2 days for each observation).\n\nThe outcome variable was substance use. The current use in this study was defined as having taken drugs at least once in the past month from the interview date.\n\nThe individual-level variables included sex, age, education, monthly income, and marital status. All variables were categorized as dichotomous variables.\n\nThe interpersonal-level variables were assessed to reflect the extent to which the drug user’s peers used methamphetamine or other IDs. Potential covariates were accounted for by adjusting for individual and interpersonal-level variables. These variables were selected on the basis of evidence regarding factors that affect substance abuse and the neighborhood environment20,21,23,25,28.\n\nThe one cluster-level variable was the level of drug use in each of the provinces sampled in 2017. The level of drug use was defined as the proportion of the population that used drugs and was partly assessed by the number of drug users who accessed the treatment system. In this study, the treatment rate was the proportion of registered clients of governmental treatment centers in the population in a given area. The estimated rate (per 1000 people) of ID users/methamphetamine users was determined from the survey in a given area in 2017.\n\nData double-entry was employed. SPSS for windows version 16.0 was used. Descriptive statistics were used to analyze all socioecological factors. Multilevel binary logistic regression analysis via generalized linear mixed models was used to analyze the associations among cluster-level variables, each covariate, and substance use. Level 2 comprised individuals at level 1 (individual-level variables and interpersonal-level variables) nested within clusters at level 2 (consisting of two variables). The model building process started with a null model consisting of no predictors, and a series of two-level models was developed. Model 1 fit only individual-level variables into the model. Then, in model 2, all interpersonal-level variables were entered into model 1. Finally, in model 3, all cluster-level variables were entered into model 2. The median odds ratio (mOR) and interval odds ratio (IOR) were applied to measure the variation of substance use in different clusters and the effects of cluster-level variables, respectively. The level of statistical significance was set to p-values < 0.05.\n\nThis research project was approved by the Human Research Ethics Committee of Khon Kaen University and was conducted in accordance with the principles of the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice standards. Written informed consent was obtained from the participants and from the parents/guardians of minors.\n\n\nResults\n\nThe median age of the agriculture-related workers was 36 years; 15.9% of the workers were <25 years old and 47.7% had obtained elementary education and lower (Table 1). One-third of the workers were poor (earned < per annum), 29.4% were current smokers and 19.1% were habitual smokers (>20 days in the past month), and 17.9% had smoked >1 pack daily. Regarding alcohol, 48.7% were current alcohol drinkers, 30.4% were habitual drinkers, and 24.1% had drunk >330 mL of beer or the equivalent amount of alcohol every time they drank (which resulted in a blood alcohol level >50 mg/dL). Regarding substance abuse, 57.7% (n=1695) of the participants currently used non-medical pharmaceutical drugs, mostly analgesics, codeine mixture cough syrup, sedatives, or opioids. Among the agriculture-related workers, 73.5% (2158) had used IDs at some point in their life, and 13.9% (408) were current ID users. This number is much higher than 1.99 in general population (Kanato et al. 2017). Current ID users were mostly regular and contracted workers also. There are a few reported histories of drug treatment for these categories of workers in the government system. All information gathered during this study is available as Underlying data32.\n\nValues are presented as number (%) or mean ± standard deviation.\n\nIDs, illicit drugs; Non-P, non-prescription drugs.\n\nAmong 408 illicit drug current users, 350 currently used methamphetamine. The rest used kratom, cannabis, inhalants, opiates/opioid, ecstasy, ketamine, and cocaine.\n\nUsers used methamphetamine 1–3 times daily (1–6 pills each time), around 30 minutes before work for first intake and every 5–6 hours booster. Mostly, users smoked methamphetamine (put 1 pill on foil then used lighter to burn and applied straw to take it). A few put 1–2 pills into drinking water. They recognized the effect of the drug (energetic, painless etc.) 1–2 minutes after smoking, or around 10 minutes after drinking. There are few reports of drug treatment for these categories of workers in the government system.\n\nConcerning both the ID use (Table 2) and methamphetamine use (Table 3), the treatment rate showed a statistically significant association with decreased substance use. Regarding ID estimation, only ID use (Table 2) was significantly associated with increased substance use; however, the effect was extremely small.\n\n1Variance at level 1 was constrained to 1. 2Estimated standard error. p < 0.05, p < 0.01, *p < 0.001.\n\nOR, odds ratio; aOR, adjusted OR; CI, confidence interval; ref, reference group; IOR, interval OR. ID, illicit drug.\n\nThe respondents who were single, had low incomes, and currently used non-medical pharmaceutical drugs were more likely to use IDs and methamphetamine (Table 2 and Table 3).\n\n1Variance at level 1 was constrained to 1. 2Estimated standard error. p < 0.05, p < 0.01, *p < 0.001.\n\nOR, odds ratio; aOR, adjusted OR; CI, confidence interval; ref, reference group; IOR, interval OR.\n\nThe mOR in all models was >1, which indicated that the between-cluster variation in substance use was greater than the within cluster-level variation; all of the IOR-80% intervals contained 1, which confirmed this finding further (Table 2 and Table 3).\n\nTable 2 presents the results of the multilevel binary logistic regression models for ID use. In model 1, the analysis of the association of the individual-level variables with substance use showed that a higher likelihood of ID use was associated with males, older age, and non-prescription drug use. In model 2, drug use among peers was not associated. In the final model (model 3), 2 cluster-level variables (i.e., treatment rate and ID estimation variables) were added to the model, and each 1-unit score increase in treatment rate was associated with a significant 12-fold increase in the likelihood of ID use.\n\nWe hypothesized that methamphetamines were used to accelerate the productivity of agriculture-related workers. A series of similar models was used to estimate the odds ratio of methamphetamine use, as shown in Table 3. In models 1 and 2 of this category, older respondents were more likely to use methamphetamines than were younger respondents. Males and non-prescription drug use were significantly associated with an increase in the likelihood of methamphetamine use. Finally, in model 3, the results showed similar associations between individual and interpersonal-level variables with substance use, as in model 2. In the final model (model 3), 2 cluster-level variables, i.e., treatment rate and ID estimation variables, were again added to the model, and each 1-unit increase in treatment rate was associated with a significant 15-fold increase in the likelihood of methamphetamine use.\n\n\nDiscussion\n\nWith the objective of exploring the prevalence of drug use among agriculture-related workers, this study found that the past month’s prevalence was much higher than the prevalence in the general Thai population, particularly for use of methamphetamine3,9. This difference in prevalence indicated that agriculture-related workers use stimulant drugs to increase their productivity, which may give them more income. Recent investigations have shown that use of IDs has spread to regular and contracted workers, indicating that IDs have become more easily accessible and affordable. Thus, functional use of drugs is acceptable by workers.\n\nThe results showed that neighborhood problems, such as health problems (treatment accessibility rate), were associated with ID use, particularly methamphetamine, consistent with other research findings22,33,34. A possible explanation for this finding is neighborhoods/clusters with a high prevalence of drug use reflect drug availability and accessibility, which could be enabling factors for workers throughout the community. Methamphetamine is an ID popularly used by drug users in Thailand. Covariates, such as sex, age, and non-prescription drug use, were related to both ID use and methamphetamine use. This finding is consistent with other research findings20,35–37.\n\nThis study used data from a cross-sectional survey; therefore, it cannot establish a temporal relationship or causality. However, the study strengths are the use of treatment rate as a cluster-level factor (enables an objective assessment), providing more reliability than that of previous subjective assessments38,39. Another study strength is it controlled for a wide range of covariates and used a large sample size and a nationally representative sample, meaning that the findings can be generally applied to the larger Thai population. Not only did this study provide further insights into cluster-level affects on substance use, but also describes associated factors adjusted for cluster environment among agricultural at national scale. However, prospective studies to further investigate may be required.\n\nThis study was successful in collecting data with a high response rate. The findings of the present study can also be applied to other similar contexts. The authors suggest further qualitative study on the beliefs of among agricultural regarding drug use for working. However, patterns of drug use may be better measured using open-ended questions, where among agricultural are asked to provide information freely without being influenced.\n\n\nConclusions\n\nThis study demonstrated association of personal characteristics of agricultural related workers and substance use particularly methamphetamine. In addition, cluster-level factors (treatment rate and ID estimation) were also significantly associated. Thus, the development of prevention measure on substance use should be taking into account the area based socioeconomic environment to pursue the more effective demand reduction.\n\n\nData availability\n\nOpen Science Framework: Drug use among agriculture-related workers in Thailand.\n\nhttps://doi.org/10.17605/OSF.IO/DWV9G32.\n\nThis project contains the following underlying data:\n\nData_Narumon.csv (demographic and questionnaire response data).\n\nData Dictionary_ Narumon .csv (data dictionary for Data_Narumon.csv).\n\nCorrect Responses_ Narumon.csv (scoring system for questionnaire responses in Data_Narumon.csv).\n\nOpen Science Framework: Drug use among agriculture-related workers in Thailand. https://doi.org/10.17605/OSF.IO/DWV9G32.\n\nThis project contains the following extended data:\n\nDrug use_QN1.jpg–Drug use_QN5.jpg (questionnaire used in this study)\n\nData are available under the terms of the Creative Commons Attribution CC 1.0 International license (CC0 1.0 Universal).", "appendix": "Competing interests\n\n\n\nN.J. and M.K. conceived the ideas; N.J. and P.L. collected the data; N.J., M.K. and P.L. analyzed the data; and M.K. led the writing.\n\n\nGrant information\n\nThis study was funded by the Office of the Narcotics Control Board (grant no. ONCB18/FY2560).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors would like to thank the ISAN Academic Network, Khon Kaen University, for their generous support. The authors would also like to thank the Office of the Narcotics Control Board for funding this study.\n\n\nReferences\n\nUnited Nations Office on Drugs and Crime (UNODC): World drug report 2018. New York: United Nations Publication; 2018. Publisher Full Text\n\nKanato M, Choomwattana C, Leyatikul P, et al.: (Eds.) ASEAN Drug Monitoring Report 2016. Bangkok: ASEAN Narcotics Cooperation Center. 2017. Reference Source\n\nKanato M, et al.: National household survey on substance use 2016. Bangkok: The Office of Narcotic Control Board (ONCB); 2017. [in Thai].\n\nThe Office of Narcotic Control Board (ONCB): Drug Situation 2559. Bangkok: The Office of Narcotic Control Board (ONCB); 2017. 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Int J Drug Policy. 2014; 25(5): 897–904. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMennis J, Stahler GJ, Mason MJ: Risky Substance Use Environments and Addiction: A New Frontier for Environmental Justice Research. Int J Environ Res Public Health. 2016; 13(6): pii: E607. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilins E, Fergusson DM, Patton GC, et al.: Adolescent substance use and educational attainment: An integrative data analysis comparing cannabis and alcohol from three Australasian cohorts. Drug Alcohol Depend. 2015; 156: 90–96. PubMed Abstract \| Publisher Full Text\n\nHadley-Ives E, Stiffman AR, Elze D, et al.: Measuring neighborhood and school environments perceptual and aggregate approaches. J Hum Behav Soc Environ. 2000; 3(1): 1–28. Publisher Full Text\n\nYangyuen S, Kanato M, Mahaweerawat U: Associations of the Neighborhood Environment With Substance Use: A Cross-sectional Investigation Among Patients in Compulsory Drug Detention Centers in Thailand. J Prev Med Public Health. 2018; 51(1): 23–32. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "60073", "date": "04 Mar 2020", "name": "Keith V. Bletzer", "expertise": [ "Reviewer Expertise Medical/cultural/social anthropology", "public health", "immigration", "alcohol/drug use among populations that experience health disparities." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, conducted from September 2017 to July 2018, considers prevalence of illicit drug use among agriculture-related workers in Thailand. The study moves past its introduction without defining the term ‘agriculture-related worker’ – how is this defined? Are these individuals working on family parcels? Do they work year-round or seasonally? Do they accept work for any crop(s) (suggesting irregular employment in fields/orchards/vineyard/etc.) or specialize in one or more crops? Do these individuals hire-out as day-labor workers? Do they work in packing and/or distribution?\n\nAre they paid by hour/by week, or do they receive wages based on piece rate (quantity they harvest)? Do they return home at the end of each day or return home at the end of short-term on-the-season employment? Do these workers live with family, do they live in dormitory barracks, do they rent living space in nearby communities, or do they live on farm premises? Defining these workers and living arrangements is important, owing to diverse types of work/employment in agriculture throughout the world. Readers outside Thailand, and agricultural labor researchers, will find valuable information in this study. In particular, how wages are determined may be related to expendable cash, utilized for drug purchase(s).\n\nData from the home survey was collected by private face-to-face interviews from September to November 2017. Each interview lasted approximately one to 1.5 hours. Were all interviewees interviewed in the same language (for the reader, name the language)? The remaining months from November 2017 to July 2018 were spent in “structured observations.” What was the purpose of these observations?\n\nResearch design was “stratified multi-stage cluster sampling” on governmental narcotic operation zones. These appear to be the provinces of Thailand. How many provinces are there in Thailand? Total sample for the survey was 2,936 persons, among whom 124 methamphetamine users participated in a structured observation (purpose?). Meth users were common among those who accessed drug treatment systems, where 80% of those treated were methamphetamine users. Also, annual prevalence was 58.73%. What types of illicit drugs are included in percentage?\n\nPage 3, first column, first paragraph: The acronym “ID use” is defined as illicit drug use, yet some readers may recognize or confuse this with ‘intravenous drug’ use. In some countries, IDU is ‘intravenous drug use.’ Usually an intravenous drug, heroin is included among identified drugs (page 3, second column, sixth paragraph) – although alternative modes of administration exist for heroin and the other drugs that are identified.\n\nPage 3, first column, second paragraph: What is meaning of “Since 1360…”?\n\nPage 3, first column, fourth paragraph: What is meaning of acronym “GDP” which refers to Thailand’s agricultural products?\n\nPage 3, second column, fourth paragraph: The total number of 5,911,567 individuals registered as agriculture-related workers – what proportion is this of the national population?\n\nPage 4, first column, third paragraph: The concept or method of “structured observation” needs description, including where this took place, what is meant by “3-5 times for each sample, 1-2 days for each observation.” Were these observations over this time, continuous? How many persons in total were observed? Importantly, what was the purpose of these observations?\n\nPage 4, second column, fifth paragraph: Drugs identified for current users differ slightly from those earlier named – including the absence of heroin (page 3, second column, sixth paragraph). “Cannabis” is the only drug named in both places – this drug also has more than one mode of administration.\n\nPage 4, Table 1: Sample size earlier identified N=2936 becomes something different, when the second column identifies n=1207 persons (807 men, 400 women), who were “not drug user.” How is this explained?\n\nAlso, why is there an age threshold (roughly 24/25 years)? This is particularly interesting, considering that “older respondents were more likely to use methamphetamines than were younger respondents” (page 6, first column, first paragraph). Were men or women more often in one or the other category of “older” and “younger,” and/or among those who use methamphetamines? Is the threshold age distinction correlated to discontinuation of drugs used earlier, which were replaced by others? For how many respondents was methamphetamine the first drug ever used? Discontinued drug(s) identified in the research, did any users return to these?\n\nPage 5, first column, first paragraph: The concept of “booster” needs to be defined, and “applied straw to take it,” needs to be described. Is this referring to oral intake, literally, OR inhalation into nostrils (nose) by means of the straw?\nDoes the “lighter to burn” refer to melting or generating a vapor for inhalation? “Drinking water” – how much: half a glass, or a full glass, or some irregular amount, based on the individual users? This information would be available from observations. Term “energetic” refers to what? Term “painless” refers to what? Is this removal and/or generating an analgesic effect that eliminates pain or a numbing predisposition for pain? Does this refer to any place within the body, or specific body sites? Most importantly, do agriculture-related workers use drugs at their work-site: field, orchard, packing plant, loading platforms, and so on?\n\nFinal sentence is unclear. Does this mean agriculture-related workers rarely seek drug treatment? Information in the final sentence does not close-out this paragraph that describes methods of use.\n\nPage 5, second column, third paragraph: A hypothesis is given, which should have been introduced earlier. Later in the manuscript, findings can be elaborated to describe whether the hypothesis was demonstrated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-818
https://f1000research.com/articles/8-817/v1	07 Jun 19	{ "type": "Research Article", "title": "Epidemiology of erectile dysfunction: A cross-sectional web-based survey conducted in an Indonesian national referral hospital", "authors": [ "Ponco Birowo", "Isaac Ardianson Deswanto", "Nur Rasyid", "Isaac Ardianson Deswanto", "Nur Rasyid" ], "abstract": "Background: Many epidemiological studies have demonstrated a high prevalence of erectile dysfunction (ED) in different parts of the world. The objective of the present study was to establish the prevalence of ED in a healthy population from Indonesia and risk factors associated with ED. Methods: We performed a cross-sectional study to estimate the prevalence of erectile dysfunction in relatively healthy males in Jakarta that aged 20 to 80 years old. This cross-sectional study utilizes a web-based survey containing a translated version of The International Index of Erectile Function (IIEF-5) in Indonesian. Sexual domain functions in the IIEF-5 include 4 domains of erectile function starting from erection confidence, erection firmness, erection maintenance and sexual satisfaction. Results: The mean age of respondents is 38.7±12.6 years old. Most of the respondents were married (77.2%), had completed or undertaking tertiary education (66.3%) and worked in privately established companies (35.7%). The prevalence of ED was 35.6% (22.3% mild, 13.7% mild to moderate, 3.1% moderate and 0.8% severe). The prevalence of ED ranges from 6.5% in the 20-29 year old group, to as high as 88.0% in respondents aged 60 years old and above. Age, hypertension, stroke, history of heart disease, diabetes, kidney disease, history of prostate operations and interpersonal stress are significantly associated with ED (p-value = <0.001, <0.001, 0.015 0.000, 0.01, 0.002, <0.001 and 0.022 respectively). Conclusion: The prevalence of ED in Indonesia is about 35.6%. The prevalence of ED in this study ranges from 6.5% to as high as 88.0%. Age, hypertension, stroke, history of heart disease, diabetes, kidney disease, history of prostate operation and interpersonal stress are significantly associated with ED.", "keywords": [ "erectile dysfunction", "epidemiology", "Indonesia", "web-based", "survey", "IIEF-5", "prevalence", "risk factors" ], "content": "Introduction\n\nErectile dysfunction (ED) is principally defined as the inability to reach and maintain an adequate erection to achieve satisfying sexual intercourse1. Although not life-threatening, erectile dysfunction has a huge impact on the quality of life of the patient as well as their sexual partners with consequential fear, depression and loss of confidence or self-esteem.\n\nMany epidemiological studies have demonstrated high prevalence of ED in different parts of the world. For instance, the Cologne Male Survey have found that the prevalence of erectile dysfunction could be as high as 19.2% amongst a group of male responder aging between 30 to 80 years old2. In the United states, about 18.4% of the adult male population (above the age of 20) suffered from sexual dysfunction3. Nikolosi et al. also explored sexual dysfunction in a number of Asian countries and found that approximately 15% of the whole Asian population suffers from ED4.\n\nA simplified version of International Index of Erectile Function (IIEF-5) is a widely used self-administered questionnaires that has been translated and validated in many different languages. This questionnaire has a high degree of specificity and sensitivity to diagnose and evaluate the prevalence of ED5. In addition, this questionnaire is also commonly used for assessing ED severity before and after appropriate therapy6–8. One limitation however, is that IIEF cannot be used to distinguish the different pathophysiology’s of ED9.\n\nTo date, the majority of studies looking at ED prevalence have focused on western countries or the Asian continent as a whole. No large population-based survey have been conducted to demonstrate the prevalence of ED in Indonesia specifically. Therefore, the objective of this present study was to establish prevalence of ED in a healthy population of males in Indonesia and risk factors for ED.\n\n\nMethods\n\nThis was a cross-sectional study used to estimate the prevalence of erectile dysfunction in relatively healthy male population in Jakarta aged 20 to 80 years old. This cross-sectional study utilized a web-based survey containing a translated version of IIEF-5 in Indonesian (see extended data10).\n\nThis study was conducted via a web-based survey in Cipto Mangunkusumo Hospital, Jakarta in March 2019. First data entry began on 1st March and last data entry was collected on 31st March 2019.\n\nThe link to the web-based survey was put on display on a banner placed in the Urology Clinic of Cipto Mangunkusumo hospital and anyone who voluntarily opened the survey through the links provided are considered as potential respondents. We did not contact potential respondents individually because it may increase the risk of selection bias. The inclusion criteria were men aged 20–80 years old who completed the questionnaire within the enrollment period. All questionnaires filled beyond the enrollment period were excluded.\n\nThis cross-sectional study has been approved by Cipto Mangunkusumo Hospital Research and Ethical Committee in February 2019 with an approval number of 0316/UN2.F1/ETIK/2018 before enrolling any participant for the study. Because this study incorporates web-based survey, written informed consents could not be obtained. Consent was taken electronically prior to participants completing the survey.\n\nWe used Google Form to provide online questionnaire and collect respondent’s data. We subsequently downloaded the completed questionnaire after the period of enrollment. Access to the completed questionnaire was considered confidential and only accessible to the authors. The online questionnaire was available here. Before being asked to fill in the questions, patients were first provided with a concise description of the study with confidentiality assurances. In order for the respondents to complete the survey, they had to provide their email address as well as their phone number to ensure that the same respondent would not take the survey more than once. We also imposed a mechanism in the survey that provided the respondents with a reminder that will pop up in their window if there are questions in the survey that have not been answered. This helps to assure that all respondents will complete all the questions provided in the survey and therefore minimizing missing data.\n\nThe translated IIEF-5 imbedded in the survey was used to determine the prevalence of. Sexual domain functions in the IIEF-5 include 4 domains of erectile function starting from erection confidence, erection firmness, erection maintenance and sexual satisfaction. These tools are useful in determining baseline erectile function as well in assess the outcome of a specific treatment modality11,12. Each IIEF-5 questions were provided with five response options, which enables the calculation of scores ranging from 5 to 25 for erectile function. The scores were then categorized into : severe ED (5–7), moderate ED (8–11), mild to moderate ED (12–16), mild ED (17–21), no ED (22–25)13 The IIEF-5 is limited by its inability to distinguish the etiologic basis for ED especially those of organic origins9. In order to address this limitation, some additional examinations should be performed, e.g. RigiScan, Doppler ultrasonography9. Unfortunately, additional examination was not possible due to the web-based nature of this study.\n\nOnly authors were able to access the completed surveys from a separate page under restricted access. Within this page, the authors are able to convert the data of the completed survey into a Microsoft Excel 2013 file which was later used for statistical analysis. This survey was planned to be maintained within one month period of time.\n\nData was analyzed using IBM SPSS Statistic 20. Demographic characteristics included in this study included the age group of respondents, marital status, educational level as well the occupation of respondents. The age group of the respondents was simplified into <40 years old and ≥ 40 years old groups to define age as a dichotomous variable to enable the subsequent analysis. Normality test using Kolmogorov-smirnov were performed for numerical data. Risk factors, including age, hypertension, stroke, history of heart disease, diabetes, kidney disease, history of prostate operation and interpersonal stress, and their association with ED in this study will be evaluated using chi-square test statistics. The result was considered significant when p < 0.05.\n\n\nResults\n\nOver 1 month a total of 255 respondents completed the web-based survey, the sociodemographic characteristics of the respondent are described in Table 1 (see underlying data14). The mean age of respondents was 38.7±12.6 years old with the majority of the respondents (31.4%) in the 30–39 years old age group. Most of the respondents were married (197/255, 77.2%) and most had completed or were undertaking tertiary education (169/225, 66.3%). The majority of respondents were employees of private companies (91/255, 35.7%).\n\nErectile function of the respondents was been stratified into ED severity categorizations based on the established IIEF-5 definitions. The prevalence and severity of ED is described in Table 2 according to age groups. The prevalence of ED in this study was 35.6% (22.3% mild, 13.7% mild to moderate, 3.1% moderate and 0.8% severe). The prevalence of ED ranged from 6.5% in the 20–29 years old age group to as high as 88.0% in respondents aged 60 years and above.\n\nThe association of ED with various potential risk factors including hypertension, stroke, history of heart disease, diabetes, kidney disease, history of prostate operation and interpersonal stress were evaluated using Chi-square analysis. The age group of the respondents was simplified into <40 years old and ≥ 40 years old groups to define age as a dichotomous variable to enable the subsequent analysis. Table 3 summarizes the findings; age, hypertension, stroke, history of heart disease, Diabetes, kidney disease, history of prostate operation and interpersonal stress are significantly associated with ED (p-value = <0.001, <0.001, 0.015, <0.001, 0.01, 0.002, <0.001 and 0.022 respectively).\n\n\nDiscussion\n\nThe prevalence of ED has been widely evaluated in many different countries around the world, however, the comparison of prevalence between countries may prove difficult ordeal due to the various questionnaires used to assess ED in different studies. Additionally, heterogeneity of age as well as ethnic distribution of different groups of respondents further complicates matters. Two previous studies by Nikolosi et al. and Akkus et al. utilized one simple question “How would you describe yourself” to assess and classify ED severity into No ED, Mild ED, Moderate ED and Severe ED15,16. Other more recent epidemiologic studies employed use of the self-reported IIEF-5 questionnaire, the most commonly used validated questionnaire that had been translated into various different languages worldwide5,17–19. Routine use of IIEF-5 in the evaluation of ED prevalence helps in gathering epidemiologic data that are comparable, possibly reproducible, and standardized facilitating cross-cultural comparison.\n\nThe discussion of sexual issues, especially those of which are related to male potency may cause embarrassment and stress. This may result in reporting bias, general underestimation or even exaggeration of sexual problems, and even the unwillingness to participate in the study overall. We aimed to address this by using a web-based survey with adaptation of IIEF-5 that any potential respondents can voluntarily access and complete without any coercions, potentially limiting any reporting bias.\n\nThe prevalence of ED in this study was 35.6% (22.3% mild, 13.7% mild to moderate, 3.1% moderate and 0.8% severe). The prevalence of ED in this study is comparably smaller to the cross-sectional study conducted by Oyelade et al. in Nigeria19 and Quilter et al. in New Zealand (58.9% and 42% respectively). This discrepancy in the prevalence rate might be explained by the difference in respondent’s age recruited in this study. In the present study 29.8% (76 out of 255) of the respondents were 20–29 years old; Oyelade et al. only recruited respondents who were 30 and above, whereas Quilter et al. only recruited respondents 40–70 years old. The prevalence of ED in this study ranges from 6.5% in the 20–29 age group to as high as 88.0% in respondents agrd 60 years old and above (Table 2). This consistent increase of ED prevalence with age was also demonstrated in a systematic review conducted by Prins et al. which found ED prevalence has been reported to be as high as 7% among men aged 18 to 29 years old, 2–9% among men aged 30–39 years old, 9–11% among men aged 40–49 years old, 16–18% among men aged 50–59 years old, 34% among men aged 60 to 69 years old, and 53% among men aged 70 to 80 years old. The rising trend in prevalence of ED as the population grows older can be explained by the presence of various comorbidities commonly found in the older population such as diabetes, heart disease, hypertension, and other vascular related diseases. These comorbidities are commonly associated with the development of ED18.\n\nIn this cross-sectional study, independent variables such as age, hypertension, history of heart disease, stroke, diabetes, kidney disease, history of prostate surgery and interpersonal stress are significantly associated with ED ((p-value = 0.000, 0.000, 0.015 0.000, 0.01, 0.002, 0.000 and 0.022 respectively). It is well known that the chances for developing ED increases with advancing age as mentioned by Prins et al. and Rhoden et al. and increases substantially from the age of 405,20. Diabetes mellitus in itself is already an independent and strong predictor for ED development as this particular medical condition is strongly involved with microvascular changes, peripheral neuropathy and endothelial dysfunction21. The significant association between history of hypertension, heart disease and interpersonal stress with ED development in this study is also consistent with the findings in a another web-based study conducted by Shaeer et al., however, they did not find any significant association of renal disease with development of ED17. This contradictive result might be caused by the ambiguous definition of renal disease used in their survey that may have confused some respondents.\n\nInterestingly, smoking which was generally thought to be associated with risk of ED, was not significantly associated with ED in the current study. This finding is consistent with studies conducted by Oyelade et al. and Shaeer et al.17,19. In contrast, an epidemiologic study conducted in Brazil by Moreira et al. found that heavy smoking (≥ 40 cigarettes/day) is a strong predicting risk factor for ED development22. The disparity in the results in between these studies is possibly due to the lack of information regarding the number of cigarettes smoked per day, as well as the duration of smoking that was not explored in this study.\n\nAlcohol consumption was not found to be significantly associated with ED, consistent with Oyelade et al. and Shaeer et al.17,19. Interestingly, Moreira et al. found that moderate alcohol consumption was inversely related with the development of ED22. Low alcohol consumption was found to improve erections and increases libido, likely due to its vasodilatory effect and suppression of anxiety. Large amounts of alcohol consumption, however, may decrease libido due to central sedation23. To correctly evaluate the effect of alcohol consumption on development of ED, the amount and duration of alcohol consumption must also be considered, which was not explored in detail in this particular study.\n\nIn summary, incorporating IIEF-5 into a carefully designed and well-structured web-based survey assists in collecting epidemiological data on the prevalence of ED that is concise, standardized, and provides easily comparable data to the surveys conducted in other countries. Additionally, web-based survey also help to spare the potential respondents from embarrassment when addressing their sexual issues, which is hoped will provide more objective responses. Lastly, the web-based survey has proven to be cost efficient and is able to cover a large sample of the population at the country level and above.\n\nOne limitation of this web-based survey, however, is the difficulty in gathering responses from individuals of lower educational status or socioeconomic level which may have restricted access to internet connection may have resulted in sampling bias. Moreover, the web link to the survey in this study was only available to those attending the urology polyclinic in Cipto Mangunkusumo hospital. Future prevalence studies of ED in Indonesia may involve the distribution of the link to this web-based survey to all health institutions in Indonesia to provide a more accurate estimation of ED prevalence in the Indonesian population.\n\n\nConclusion\n\nIn conclusion, the prevalence of ED in our study was 35.6% (22.3% mild, 13.7% mild to moderate, 3.1% moderate and 0.8% severe). The prevalence of ED in this study ranges from 6.5% in the 20–29 years old group to 88.0% in respondents aged 60 years old and above. This shows the consistent increase in the prevalence of ED as the population is ages. Additionally, independent variables such as age, hypertension, history of heart disease, stroke, diabetes, kidney disease, prostate operation and interpersonal stress are significantly associated with ED.\n\n\nData availability\n\nHarvard Dataverse: ED Web-based Survey. https://doi.org/10.7910/DVN/PEX2YB14\n\nThis project contains the following underlying data:\n\nDE-revised.tab (Extracted web survey data)\n\nLink provided to participants: https://docs.google.com/forms/d/e/1FAIpQLSfYGnSIsnY3-kgGH_50Fe_DdNgTMnkieeBFMaNLkgPnnJbRTw/viewform\n\nHarvard Dataverse Repository: Questionnaire ED (EN). https://doi.org/10.7910/DVN/TAFOCB10\n\nThis project contains the following extended data:\n\nQuestionnaire ED_ID.docx (Study questionnaire, Indonesian)\n\nQuestionnaire ED_revised.docx (Study questionnaire, English)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNIH Consensus Conference. Impotence. NIH Consensus Development Panel on Impotence. JAMA. 1993; 270(1): 83–90. PubMed Abstract \| Publisher Full Text\n\nBraun M, Wassmer G, Klotz T, et al.: Epidemiology of erectile dysfunction: results of the 'Cologne Male Survey'. Int J Impot Res. 2000; 12(6): 305–11. PubMed Abstract \| Publisher Full Text\n\nSelvin E, Burnett AL, Platz EA: Prevalence and risk factors for erectile dysfunction in the US. Am J Med. 2007; 120(2): 151–7. PubMed Abstract \| Publisher Full Text\n\nNicolosi A, Glasser DB, Kim SC, et al.: Sexual behaviour and dysfunction and help-seeking patterns in adults aged 40-80 years in the urban population of Asian countries. BJU Int. 2005; 95(4): 609–14. PubMed Abstract \| Publisher Full Text\n\nRhoden EL, Telöken C, Sogari PR, et al.: The use of the simplified International Index of Erectile Function (IIEF-5) as a diagnostic tool to study the prevalence of erectile dysfunction. Int J Impot Res. 2002; 14(4): 245–50. PubMed Abstract \| Publisher Full Text\n\nPorst H, McVary KT, Montorsi F, et al.: Effects of once-daily tadalafil on erectile function in men with erectile dysfunction and signs and symptoms of benign prostatic hyperplasia. Eur Urol. 2009; 56(4): 727–35. PubMed Abstract \| Publisher Full Text\n\nPorst H, Giuliano F, Glina S, et al.: Evaluation of the efficacy and safety of once-a-day dosing of tadalafil 5mg and 10mg in the treatment of erectile dysfunction: results of a multicenter, randomized, double-blind, placebo-controlled trial. Eur Urol. 2006; 50(2): 351–9. PubMed Abstract \| Publisher Full Text\n\nSkoumal R, Chen J, Kula K, et al.: Efficacy and treatment satisfaction with on-demand tadalafil (Cialis) in men with erectile dysfunction. Eur Urol. 2004; 46(3): 362–9; discussion 369. PubMed Abstract \| Publisher Full Text\n\nTang Z, Li D, Zhang X, et al.: Comparison of the simplified International Index of Erectile Function (IIEF-5) in patients of erectile dysfunction with different pathophysiologies. BMC Urol. 2014; 14: 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeswanto IA: Questionnaire ED (EN). Harvard Dataverse, V2. 2019. http://www.doi.org/10.7910/DVN/TAFOCB\n\nBella AJ LT: Male Sexual Dysfunction. 17th ed. Tanagho EA MJW, editor. McGrawHill Medical: 2008; 589–610.\n\nMelman A, Fogarty J, Hafron J: Can self-administered questionnaires supplant objective testing of erectile function? A comparison between the International Index Of Erectile Function and objective studies. Int J Impot Res. 2006; 18(2): 126–9. PubMed Abstract \| Publisher Full Text\n\nRosen RC, Cappelleri JC, Gendrano N 3rd: The International Index of Erectile Function (IIEF): a state-of-the-science review. Int J Impot Res. 2002; 14(4): 226–44. PubMed Abstract \| Publisher Full Text\n\nDeswanto IA: ED Web-based Survey. Harvard Dataverse, V2. 2019. http://www.doi.org/10.7910/DVN/PEX2YB\n\nNicolosi A, Moreira ED Jr, Shirai M, et al.: Epidemiology of erectile dysfunction in four countries: cross-national study of the prevalence and correlates of erectile dysfunction. Urology. 2003; 61(1): 201–6. PubMed Abstract \| Publisher Full Text\n\nAkkus E, Kadioglu A, Esen A, et al.: Prevalence and correlates of erectile dysfunction in Turkey: a population-based study. Eur Urol. 2002; 41(3): 298–304. PubMed Abstract \| Publisher Full Text\n\nShaeer O, Shaeer K: The Global Online Sexuality Survey (GOSS): the United States of America in 2011. Chapter I: erectile dysfunction among English-speakers. J Sex Med. 2012; 9(12): 3018–27. PubMed Abstract \| Publisher Full Text\n\nQuilter M, Hodges L, Hurst P Von, et al.: Male Sexual Function in New Zealand: A Population-Based Cross-Sectional Survey of the Prevalence of Erectile Dysfunction in Men Aged 40-70 Years. J Sex Med. 2017; 14(7): 928–36. PubMed Abstract \| Publisher Full Text\n\nOyelade BO, Jemilohun AC, Aderibigbe SA: Prevalence of erectile dysfunction and possible risk factors among men of South-Western Nigeria: a population based study. Pan Afr Med J. 2016; 24: 124. PubMed Abstract \| Free Full Text\n\nPrins J, Blanker MH, Bohnen AM, et al.: Prevalence of erectile dysfunction: a systematic review of population-based studies. Int J Impot Res. 2002; 14(6): 422–32. PubMed Abstract \| Publisher Full Text\n\nGoyal A, Singh P, Ahuja A: Prevalence and Severity of Erectile Dysfunction as Assessed by IIEF-5 in North Indian Type 2 Diabetic Males and Its Correlation with Variables. J Clin Diagnostic Res. 2013; 7(12): 2936–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreira ED Jr, Bestane WJ, Bartolo EB, et al.: Prevalence and determinants of erectile dysfunction in Santos, southeastern Brazil. Sao Paulo Med J. 2002; 120(2): 49–54. PubMed Abstract \| Publisher Full Text\n\nMiller NS, Gold MS: The human sexual response and alcohol and drugs. J Subst Abuse Treat. 1988; 5(3): 171–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49655", "date": "13 Jun 2019", "name": "Hong-Jun Li", "expertise": [ "Reviewer Expertise Andrology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the current study, the authors tried to estimate the prevalence of ED in Indonesia population through a web-based survey. I have several concerns about the methods and results parts.\n\nMajor points: First, compared with traditional studies using face-to-face questionnaires, there are several limitations must be stressed concerning web-based survey:\nWeb-based survey cannot guarantee that all the terms are correctly understood by the participants. Usually an experienced physician is necessary for explanation of the questions in the study. Physical examination cannot be performed. Some physical examination results such as testicular volume, varicocele... may be associated with ED. Additionally, detection of sexual hormones (serum testosterone, estrogen, prolactin) is also required for a cross-sectional study researching ED. Absence of these will result in potential bias. According to the description in Methods part, the link to the web-based survey was put on display on a banner placed in the Urology Clinic. I doubt whether these participants can represent general population in Indonesia? These participants (I think most of them were patients in the Urology Clinic) were those who were interested about this issue. And this will result in selection bias. I am wondering whether there were some participants completing the web-survey directly through searching in the Internet?\nSecond, the sample size was not predetermined. I don’t think the sample size (255) is large enough to estimate the prevalence of ED in Indonesia. The authors need to address the end-points of their study.\n\nThird, concerning statistics, a multivariate analysis may be performed to get a comprehensive understanding of risk factors of ED.\n\nFourth, some factors were not listed in the questionnaire, which were commonly used in ED research, such as previous diagnosed of CP/CPPS, anxiety, depression...\n\nMinor points: In the abstract-methods: what is the definition of “relatively healthy males”?\n\nScientific paper is not easy to accept unclear words, such as “relatively”, and “about 35.6%” in abstract-conclusion part.\n\nReferences were not new and required to be up-dated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "95368", "date": "11 Oct 2021", "name": "Ahmed Hosny", "expertise": [ "Reviewer Expertise Andrology and STDs" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 population-based survey to demonstrate the prevalence of ED in Indonesian healthy males and the risk factors for ED.\nI have the following comments:\nFor your sample size - did you do a power calculation? I think that this sample size is small to address the prevalence of a disease.\n\nAdding the age range up to 80 years, I think that affects the total prevalence of the disease.\n\nThe authors address the risk factors based only on the online questionaries with no attached data to confirm it, which makes this more subjective and only patient-based.\n\nWhat is the criteria or the definition they judge that the participant is healthy?\n\nThe authors mentioned in the risk factors “hormonal disease”, I think it needs to be more specified.\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": [] } ]	1	https://f1000research.com/articles/8-817
https://f1000research.com/articles/8-808/v1	06 Jun 19	{ "type": "Research Article", "title": "Prevalence and risk factors for low back pain among university teaching staff in Nairobi, Kenya: a cross-sectional study", "authors": [ "Saikou Yaya Kollet Diallo", "Marshal M. Mweu", "Simeon Ochanda Mbuya", "Mutuku Alexander Mwanthi", "Marshal M. Mweu", "Simeon Ochanda Mbuya", "Mutuku Alexander Mwanthi" ], "abstract": "Background: To date, there are few studies carried out on low back pain (LBP) among university teaching staff in developing countries despite academics being a high-risk group for LBP. In Kenya, to the best of our knowledge, there are no published studies that have investigated risk factors for LBP among teaching staff. The objectives of this study were to estimate the prevalence of LBP among teaching staff of the University of Nairobi (UoN), during the period June 2016 – May 2017, and to identify its socio-demographic and work-related risk factors. Methods: An analytical cross-sectional study design was used to estimate the prevalence and investigate the risk factors for LBP among 136 teaching staff of UoN. A semi-structured questionnaire was used to collect data on LBP history, work-related and socio-demographic characteristics of the study participants. The 12-month prevalence of LBP and its associated 95% exact binomial confidence interval were estimated. A mixed-effects logistic regression model was used to evaluate the relationship between the predictors and LBP. Results: The estimated 12-month prevalence of LBP was 64% (95% CI: 55.3%–72.0%). From the multivariable analysis, physical inactivity (aOR: 6.0; 95% CI: 1.2–29.6), office chairs without lumbar supports (aOR: 3.3; 95% CI: 0.1–0.9) and high workplace stress (aOR: 4.4; 95% CI: 1.1–17.5) were identified as significant risk factors for LBP among the respondents. Conclusions: This study has revealed a high burden of LBP among teaching staff of the UoN and undoubtedly mimics the situation in other higher learning institutions in Kenya. Physical inactivity, sitting on chairs without lumbar supports and workplace stress have been identified as modifiable risk factors for LBP among teaching staff. This suggests a need to strengthen advocacy for regular physical activity, team-building activities and investment in office infrastructure to mitigate the effects of LBP within learning institutions.", "keywords": [ "low back pain", "prevalence", "risk factors", "University teaching staff." ], "content": "1. Introduction\n\nDisorders of the musculoskeletal system (MSDs) constitute the second most common cause of disability worldwide – accounting for 169,624,000 disability-adjusted life years (DALYs) as of 2010 which is a 45.5% increase over 10 years1,2. Of all work-related MSDs, low back pain (LBP) remains the most frequently diagnosed condition since the low back vertebral discs are subject to the greatest mechanical stress, compression force and degenerative changes3–5. LBP is defined as pain localised between the lower margin of the twelfth ribs and the lower gluteal folds with or without leg pain that lasts at least one day6,7.\n\nLBP was ranked as the first contributing factor to global disability out of 291 conditions investigated in 2010 and the third in Eastern Sub-Saharan Africa, measured in years lived with disability7. LBP prevalence was found to be 50% among physicians and dentists in India5, 61.9% among Ugandan nurses4 and 77.2% in theatre nurses in Nigeria3. It is estimated that more than 80% of people end up suffering from LBP at some point in their lifetimes8. Only 5–15% of LBP cases have a specific cause such as an osteoporotic fracture, neoplasm or infection9.\n\nLBP arises from several contributing factors, namely: socio-demographic, ergonomic and psychosocial predictors10. Low back injuries leading to LBP are associated with occupational risk factors, with 11% to 80% of them being attributable to ergonomic factors such as prolonged sitting, lifting, bending and twisting10–13. Psychosocial factors account for 14% to 63% of low back injuries, mainly high job demands, job dissatisfaction and stress at the workplace10–12,14. Socio-demographic factors equally play an important role in LBP occurrence and comprise both individual and lifestyle factors. Of these, the most commonly identified are lack of physical exercise, old age, female gender, obesity and smoking11–15.\n\nThe job description for teachers comprises a broad range of duties and responsibilities which may predispose teachers to LBP. For instance, while preparing teaching materials, teachers may experience prolonged sitting either in the office or at home. When delivering lectures, they may be upstanding for long hours, or may adopt awkward postures like bending, reaching and twisting. They may have to use inappropriate furniture such as immobile chairs without back support and non-mechanized tables. These varying postures may trigger back pain owing to the continuous loading of back muscles12,16,17. In Kenya, little has been published on LBP. However, the few available studies showed high prevalence of the condition in Nairobi: 76.5% among sedentary office workers18 and 90.5% among hospital employees19. With a considerable proportion of teaching staff in Kenya being past the age of 50 years, it is anticipated that the magnitude of LBP would be high with attendant productivity losses and financial burden to the University community.\n\nThe objectives of this study were to estimate the prevalence of LBP among the teaching staff of the College of Health Sciences, University of Nairobi, during the period June 2016-May 2017, and to identify its socio-demographic and work-related risk factors with a view to informing the formulation of effective prevention and control strategies for LBP within teaching institutions in Kenya.\n\n\n2. Methods\n\nThe study was conducted at the University of Nairobi (UoN), College of Health Sciences (CHS) – one of the six constituent colleges of the UoN. Notably, UoN is the largest higher learning institution in Kenya, whose working conditions closely mimic those of other public tertiary institutions in the country. The CHS consists of five schools (Medicine [SOM], Dental Sciences [SDS], Pharmacy [SOPharm], Nursing Sciences [SON], and Public Health [SPH]) and four institutes (Tropical and Infectious Diseases [UNITID], Kenya AIDS Vaccine Initiative [KAVI], East African Kidney Institute [EAKI] and the Centre for HIV Prevention and Research [CHIVPR]).\n\nAn analytical cross-sectional study design was employed to estimate the prevalence and investigate the risk factors for LBP among the college teaching staff of UoN from June 2016 to May 2017. The study was reported as per the STROBE guidelines for reporting observational studies20.\n\nThe study population consisted of all teaching staff of the CHS eligible to participate in the study. To be eligible for participation, a staff member had to have been employed for at least 12 months prior to the commencement date of the study (May 2017) and have given informed written consent for participation. Moreover, those having LBP due to trauma, infection or tumour were excluded from the study. The sampling frame of teaching staff was secured from the college registry. To obtain the study sample, a stratified random sampling technique (with strata being the constituent Schools and Institutes of the CHS) was used. Within each stratum, a simple random sample was selected, such that the number sampled per stratum was proportional to the size of the stratum. Arguably, stratified random sampling ensures that all strata are represented in the sample and further improves precision of the estimates by removing the between-strata variation21. A flow chart of the sampling strategy is shown in Figure 1.\n\nAn LBP case was defined as a staff member who had a history of pain localised in the lower back (as previously defined) lasting for at least 24 hours within the 12-month study period, had been physically examined by a physician at a health facility and further undergone a diagnostic imaging examination revealing lumbar disc degeneration. Contrastingly, a non-case was a study participant without a previous history of LBP within the same study period.\n\nThe required sample size was determined as specified by Kelsey, JL et al.22 for cross-sectional studies:\n\n\n\n\n\n\n\nWhere:\n\nn1 is the number of cases and n2 is the number of non-cases; p1 is the proportion of individuals who did not exercise and had LBP; p2 is the proportion of individuals who exercised and had LBP – estimated to be 43.1% based on a previous study14. Notably, Zα/2 (1.96) and Zβ (-0.84) are the values which specify the desired 2-tailed confidence level (95%) and statistical power (80%) respectively. The odds ratio (OR) for the effect of the primary exposure (lack of physical exercise) was hypothesised to be 2.214. The ratio (r) of unexposed to exposed individuals was set at 1. Given these figures, a total sample size of 204 participants was derived.\n\nInitially, two research assistants were recruited and trained to aid with the data collection exercise that spanned a two-month period May 31st–July 31st 2017. As for the data collection, a semi-structured questionnaire (see extended data23) was administered to the study participants capturing details of their LBP history, and predictors: work-related (length of working day, office chair design, stress, social support and job satisfaction) and socio-demographic characteristics (age, sex, marital status, body mass index (BMI), level of education, school (including department), physical activity engagement, tobacco use and level of alcohol intake). The predictors were assessed as given in Table 1. A conceptual framework depicting the predictor-outcome relationship is displayed in Figure 2.\n\nThe study respondents provided written informed consent expressing their willingness to take part in the study. Approval for the study was granted by the Kenyatta National Hospital and University of Nairobi joint Ethics and Research Committee (KNH-ERC/A/171).\n\nGranted that cross-sectional studies are prone to a range of biases that may invalidate study results, deliberate attempts were made to minimize their occurrence. Ergonomic and lifestyle factors are readily modified once individuals are diagnosed with LBP. As such, to reduce the possibility of reverse causality involving these set of factors, specific questions targeted the period preceding the onset of symptoms characteristic of LBP for case respondents. To standardise the interview process and thus minimize interviewer bias, the research assistants were trained on sound interviewing techniques. As non-response may introduce selection bias in cross-sectional studies, non-responders were aggressively followed up with reminders to achieve a reasonable response rate.\n\nPrior to data entry, questionnaire responses capturing qualitative variables were coded. The data were then double-entered in an EpiData v3.1 spreadsheet by two independent data entry clerks to minimize errors. The validated dataset was then exported to Stata v13 software for data cleaning and analyses. Continuous variables were summarized using the median and inter-quartile range (IQR) as well as histograms and boxplots. For categorical variables, proportions were computed. The prevalence of LBP and its associated 95% exact binomial confidence interval were estimated. Code for analysis is available as extended data25.\n\nFor univariable analyses, a mixed-effects logistic regression model was used to evaluate the effect of each predictor on LBP, with the variable department included as a random effect to account for clustering of the outcome within departments. The significance of each of the predictors at this stage was evaluated at a liberal P≤0.20. As inclusion of age, BMI and length of working day as continuous predictors in the univariable models yielded insignificant results, these were categorised and reassessed for significance. In particular, age was grouped into three categories: ≤43yrs; 44–57yrs; ≥58yrs, BMI was classified into the four BMI categories26: Underweight(<18.5), Normal weight (18.5–24.9), Overweight (25.0–29.9) or Obese (≥ 30.0) and length of working day was categorised into: ≤8hrs or >8hrs.\n\nVariables that were found to be significant in the univariable analyses were then offered to a multivariable model where a backward step-wise approach was used to eliminate variables at P≥0.05. To minimize confounding, elimination of non-significant predictors was only considered when their exclusion from the model did not result in a more than 30% change in the effects of the remaining variables21. Two-way interactions were fitted between the remaining variables of the final model and assessed for significance.\n\n\n3. Results\n\nA total of 204 teaching staff of the CHS were invited to participate in the study, from whom 167 consented to participating in the survey, giving a response rate of 81.9%. However, of the 167 participants, 31 were excluded from the analyses for reporting trauma and/or infection as the reason(s) for their back pain. Therefore, 136 participants were considered in the analyses [see underlying data].\n\nDescriptive statistics for the predictors of LBP are displayed in Table 2. Notably, the median age for the participants was 51 years (Range: 31–81yrs). A typical working day was 10 hours long (range: 4–18hrs). Only 44.9% (n=61) of the participants regularly exercised. Participants with office chairs that had lumbar support represented 41.9% (n=57) of the total. The estimated 12-month period prevalence of LBP was 64% (95% CI: 55.3%–72.0%).\n\nBased on results of the univariable analyses, the variables: sex, age, school, physical exercise, office chair design and level of workplace stress, were significantly associated with LBP at P 0.20 (Table 3). These were subsequently offered to the multivariable model. In the multivariable analysis, only physical exercise, office chair design and level of workplace stress were shown to be significant predictors of LBP at the 5% of significance level (Table 4).\n\na, b, c, d, e, f Variables eligible for inclusion in the multivariable model (P≤0.20)\n\naAdjusted odds ratio\n\nCompared to respondents who regularly exercised, participants who rarely and never exercised had respectively about three (aOR: 2.8; 95% CI: 0.9–8.4) and six times (aOR: 6.0; 95% CI: 1.2–29.6) the odds of LBP controlling for their office chair design and workplace stress level. Participants who sat on chairs with lumbar support had a third (aOR: 0.3; 95% CI: 0.1–0.9) the odds of LBP as those who did not regardless of their level of physical activity and stress at their workplace. Irrespective of their level of physical activity and design of their office chair, respondents who experienced high and low stress levels at their workplace had roughly four times (aOR: 4.4; 95% CI: 1.1–17.5) and three-fifths (aOR: 0.6; 95% CI: 0.2–1.9) the odds of LBP respectively, as those whose perception of stress was medium.\n\n\n4. Discussion\n\nThe prevalence of LBP among teaching staff of the CHS, UoN was estimated to be 64.0%. This is a higher prevalence than demonstrated by most studies conducted among teachers in which LBP prevalence ranged between 22.3% (Thailand) and 57.5% (Ethiopia)12–15. This variation could be attributable to age differences between the study participants, with those in the mentioned studies being on average younger (mean age: 34.7–38yrs) than those included in the present study (mean age: 50.9yrs). An age-LBP association has been demonstrated, with LBP being more prevalent among individuals over 40 years12,14,27.\n\nThis study has shown that teachers who either do not exercise or do so infrequently, have higher odds of experiencing LBP than their counterparts who exercise regularly. This finding is consistent with study observations made in Israel, Iran, India, South Korea and Ethiopia11,14,15. Regular physical exercise has been shown to strengthen lower back muscles and maintain the spine in proper alignment for optimal function. Furthermore, routine exercises increase blood supply to the spine muscles, joints and intervertebral discs minimizing injury and enhancing their repair14,28,29. It has been suggested that a minimum of 30 minutes of regular exercise could increase trunk flexibility and stimulate an adequate production of endorphins that could diminish pain sensation30,31.\n\nSitting on a chair with back support had the effect of lowering the odds of LBP. The use of lumbar supports has been widely advocated because of their well-known function of preserving the integrity of the low back curves, thus reducing the risk of LBP32–34. Additionally, the tilt of the lumbar support permits the person using it to sit with his/her upper body slightly reclined which ensures proper body weight distribution32,35,36.\n\nThere was a noticeable association between perceived stress levels at the workplace and the reporting of LBP. High stress levels have been associated with the stimulation of the sympathetic nervous system prompting the release of stress mediators that can strain the musculoskeletal system resulting in LBP. Our finding concurs with that reported by an Ethiopian study in which participants reporting stress had roughly two times the odds of experiencing LBP than those without stress14. Similar associations have also been observed elsewhere11,28.\n\nOur study did not suggest any evidence for the existence of a real difference in LBP prevalence between sexes. This could be partly ascribable to our study participants being generally older and hence exposed to a similar risk. Nevertheless, among younger participants, being female has been associated with an elevated risk of LBP owing to hormonal imbalances11,14,37. More so, during pregnancy, hormonal changes responsible for loosening the spinal ligaments coupled with the extra weight that stresses the lower back muscles heighten the risk of LBP38,39.\n\nTaking into account other study variables, age did not emerge as a significant predictor for LBP in the present study. A likely explanation for this would be that older participants aware of their disproportionately higher risk, engaged themselves in regular exercises at a comparably higher frequency (as per the data: P=0.02) thus arguably, balancing out their LBP risk to that of their younger counterparts. Nonetheless, a number of studies have reported age as a significant risk factor for LBP; old age being associated with spine and vertebral disc degeneration as well as loss of connective tissue elasticity that can result in LBP12,14,29.\n\nWorking at a particular school did not significantly influence a participant’s likelihood of LBP. This is conceivable considering that the respondents are likely to have similar work responsibilities entailing preparation of teaching materials, lecturing, grading of students’ papers, mentoring and supervision that often demand extended periods of sitting and standing. This prevalence homogeneity may also denote uniformity in the distribution of office furniture designs across schools.\n\nA couple of limitations are inherent in the present study. Definition of the outcome was pegged on self-report which could have introduced non-differential misclassification with a potential to bias the estimated odds ratios towards null. As measurement of exposures relied on recall which could have been incomplete especially for chronic cases, this would have the potential of biasing the effect estimates. Since non-responders tend to systematically differ from responders with regards to a range of health outcomes, it is anticipated that the current study’s prevalence underestimates the true burden of LBP in this study population. In light of the above-mentioned limitations, it should be borne in mind that the results of this study are merely hypothesis generators and hence, stronger study designs such as case-control or cohort studies are recommended to validate the findings.\n\n\n5. Conclusions\n\nThis study has revealed a high burden of LBP among teaching staff of the University of Nairobi and undoubtedly mimics the situation in other higher learning institutions in Kenya. Lack of physical activity, seating on chairs without lumbar support and workplace stress have been identified as modifiable risk factors for LBP among teaching staff. Considering the similarity in demographics and working conditions across public institutions in Kenya, these findings are readily generalisable to other public tertiary institutions within the country. Consequently, there is a pressing need for university managements to: (1) invest in suitable office furniture, in particular, office chairs fitted with appropriate lumbar supports and (2) raise advocacy for and facilitate the implementation of regular workouts and departmental team-building activities with a view to mitigating the burden of LBP among their staff.\n\n\n6. Data availability\n\nThe raw dataset for the study is kept under restricted access since it contains sensitive participant information. Access to the raw data is possible upon placing a formal request to the corresponding author (disykgn@gmail.com). The replication data, analysis script and questionnaire for this manuscript are available from figshare.\n\nFigshare: LBP_UoN_CHS_2.dta. https://doi.org/10.6084/m9.figshare.8148779.v140\n\nThis project contains the following underlying data:\n\nLBP_UoN_CHS_2.dta (Low back pain survey data)\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: LBP Study Questionnaire_UoN. https://doi.org/10.6084/m9.figshare.8197598.v123\n\nThis project contains the following extended data:\n\nQuestionnqire_LBP Study among teaching staff UoN, Kenya.pdf (Low back pain survey questionnaire)\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: LBP study_CHS_UoN_Stata code. https://doi.org/10.6084/m9.figshare.8197715.v225\n\nThis project contains the following extended data:\n\nLBP study_Kenya_UoN_CHS_Stata Code.do (Low back pain Stata code)\n\nData are available under the terms of the MIT licence.", "appendix": "Grant information\n\nThis work was supported by the Association of African Universities [PC/6].\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\nThe authors wish to express their sincere gratitude to the study participants for contributing to the research.\n\n\nReferences\n\nInstitute for Health Metrics and Evaluation: The Global Burden of Disease: generating evidence, guiding policy. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGordon R, Bloxham S: A Systematic Review of the Effects of Exercise and Physical Activity on Non-Specific Chronic Low Back Pain. Healthcare (Basel). 2016; 4(2): pii: E22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHedge A: Ergonomic seating. Cornell University of Education; 2013. Reference Source\n\nHermanMiller Inc. Body support in the office: Sitting, seating and low back pain. HermanMiller Inc; 2002. Reference Source\n\nLoewenstein H, Pateterson I, Shuke J: Comparison of lumbar curves, when sitting on Nada-chair, sitting on a conventional chair and standing. Beaver College. 1989. Reference Source\n\nLueder R: Ergonomics of Seated movement: A review of the scientific literature. Ergonomic Review of the research. 2004. Reference Source\n\nWorksafeNB: Office ergonomics: Guidelines for preventing Musculoskeletal injuries. 2017 [accessed on 30th September, 2017]. 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Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8148779.v1" }	[ { "id": "59230", "date": "24 Feb 2020", "name": "Marcus Fraga Vieira", "expertise": [ "Reviewer Expertise Biomechanics", "gait and posture", "pattern recognition", "feature extraction", "feature selection", "feature classification." ], "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 deals with the prevalence of low back pain in teachers at the University of Nairobi and analyzes the relationship between the incidence of low back pain and some variables taken as risk factors.\nIn general, the manuscript was well written, and the study was well conducted, with a consistent statistical analysis and a good discussion.\nI only have a few minor comments and suggestions.\n\nPage 4 - Sample size:\nUnfortunately, the calculated sample size could not be guaranteed. Given the possibility of sample loss in most studies, why did the authors not start the study with a larger sample than that calculated here?\n\nPage 4 - Study variables:\nI think it would be interesting if the authors could justify the choice of the study variables/factors analyzed here. I think some other factors are, in some extension, more important than that cited here.\n\nFor example: office chair design is important, but I think the number of hours of sitting work is more or as important as office chair design. Some comments about this would be welcome. Something like \"teachers spend most of their time working in a sitting position, thus we considered the chair design as a risk factor for LBP\", for example.\n\nHowever, a question can be raised: why not evaluate the number of hours of sitting work?\n\nConceptual framework in Figure 2:\nI recommend emphasizing that those relationships presented in Figure 2 are a conception of the authors that motivate the choice of the \"risk factors\" presented here.\n\nThose relationships may be different in other conditions, or for other populations, or even other authors may interpret those relationships in a different way.\n\nPage 5 - Figure 2 captions:\nI recommend not using this term here: \"causal diagram\". We cannot say that those factors are causal factors of LBP: the cause-effect relationship between them and LBP cannot be guaranteed. Furthermore, the relationships presented here are a personal interpretation of the authors. Other authors may establish a different relationship between the factors presented here. For example: for the authors, the level of stress is a risk factor for LBP, but, perhaps, LBP increases the level of stress, especially for a teacher who works seated most of the time.\n\nPage 6 - 4.2 Risk factors for LBP:\nI suggest changing this subsection to something such as \"Variables studied as risk factors for LBP\".\n\nThere are other variables that may be related to LBP, and some variables studied here may not be considered risk factors for LBP by other authors.\n\nPage 7 - Last paragraph:\nThe interpretation here is somewhat speculative. However, the level of physical activity for each age group can be verified. The authors have this information.\n\nThis information would be welcome and would support the interpretation raised in that paragraph.\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": "58991", "date": "02 Mar 2020", "name": "Peter R. Croft", "expertise": [ "Reviewer Expertise Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is good to see original studies on low back pain (LBP) from Africa. LBP is a global problem and yet is under-represented in research from countries such as Kenya.\nThis is a generally well-written paper, with appropriate methods and analysis and presentation of results. So the overall opinion is that this is a useful, well-performed and reasonably well-reported study.\n\nThere are some weaknesses however, and they are discussed below in the hope that they will help interpretation of results and be constructive for future research by the authors.\n1. Study rationale: The authors carefully chose a narrow focus for their paper. This study is about LBP in University teaching staff, and the limit of the generalisations made at the end of the paper is that results can be extended to teaching staff in other Higher Education institutions in Kenya. The authors’ main conclusions relate to a proposal that the risk factors for low back pain identified in the study should and could be reduced in these workplaces. This is admirable as a stimulus to local preventive action, but the interest for an international readership would be (a) to consider to what extent the prevalence estimate in this workplace population represents the occurrence of LBP in older Kenyans and Africans more widely, and (b) to know how the findings on the risk factors for LBP compare with and contribute to the extensive global literature on these factors. Such wider objectives would draw out the study’s importance beyond the local occupational setting, but require a more detailed discussion section (see below).\n2. Study context: It is rather alarming to read that this observational study of LBP in a random sample of teachers in a University health faculty could take as its case definition low back pain of minimum 24 hours plus visit to health care plus spinal imaging. Given that international guidelines recommend strongly against X-ray for uncomplicated back pain, does this mean that in Kenya most people experiencing back pain will expect to have an X-ray (which would be important contextual information for the paper) OR does it mean that this is a highly selected sample of all those in this population who had LBP (i.e. were there many people reporting back pain who had not had an X-ray and were therefore excluded from the study? – this would be important for study interpretation).\n3. Study style: The authors have appropriately and importantly included workplace stress and supervisor support and demographic/lifestyle factors among the potential risk factors for LBP. This is excellent, but much of the introduction and discussion defaults to “physical” explanations of, and mechanisms for, LBP. Chronic LBP is a complex biopsychosocial phenomenon, in which mental health and cultural perceptions play as big a role as mechanical factors, and this could be recognised more explicitly in the introduction and discussion sections.\n4. The definition of low back pain: The definition of the area of the back to be included is admirably clear, as is the minimum duration of 24 hours. However it is unfortunate (especially for the estimate of prevalence) that there were no further details on the pain itself and its impact, in order to sub-group the prevalence estimate according to severity. Back pain is so common that, to provide prevalence estimates that are more widely useful for planning, prevention and care, it should be standard practice in prevalence surveys to add a measure of severity and impact, such as that in Von Korff et al, Pain, 2020.1 (The authors might argue that this was not so necessary for the risk factor component of their study but that is one reason for clearly separating the discussion of prevalence and risk factor components of the study – see comments below).\n5. Study design: The authors say at the end of their paper that more research with stronger designs (including case-control) is needed to confirm the results found in their cross-sectional study. I think the authors are underplaying their own design. There is an argument for saying that their study has two components – first, a cross-sectional population survey to estimate prevalence, and second, a population-based case-control study to study associations with LBP. The arguments for suggesting that the risk factor study is a case-control study are:\nExposures were compared between those without LBP in the survey who represent a population sample of “controls”, and “cases” with LBP (after exclusion of cases with other causes (injury/infection)).\n\nThe sample size has been estimated on the basis of exposure data, as for a case-control study, rather than for the prevalence study.\n\nThe authors have attempted to ensure that, when gathering exposure data, participants were asked to recall exposures in periods prior to the onset of their LBP – a classic piece of case-control design.\n\nIf these two components of their study were separated, then the authors could usefully critically reflect on the following design points:\na. It was good to see a flow diagram of response and exclusion, and a high response rate, for the prevalence study. But what was the size of the total eligible population that provided the basis for stratified sampling for the study population (before exclusions, invitations, and nonresponse)? A small point to note is that the word “aggressive” in relation to following up non-responders is best avoided. It is better to give the details of exactly what was done, including a clear account of how and when people’s non-response is taken as final.\nb. What was the origin of the questions used in the semi-structured interviews, and had there been any investigation of questionnaire validity prior to the main study? For example, alternative external records on the “lumbar support” seating data might have been sought from departmental purchasing data; repeatability of physical activity recall might have been tested by repeat interviews in a sub-sample of participants; were the interviewers aware of the participants’ back pain status when they asked about the risk exposures? Even if these “safeguards” were not carried out, the authors could consider adding a critical discussion about what they might have added to the credibility and validity of the study.\n6, Study analysis:\nIndependent data entry is excellent practice – but some details of how any disagreements were handled would be useful, together with a summary of such disagreements.\n\nMedians are fine but it might be better to make a statement about distributions of the relevant continuous variables because looking at them it is unclear why means and standard deviations were not chosen. The authors seem a little uncertain themselves because mean age is quoted in the discussion and median age in methods and results.\n\nThe analysis of results was nicely structured and followed a clear and appropriate pathway from descriptive to univariable to backwards multivariable models. However there was often a slightly ‘formulaic’ style to the writing of this, which could be avoided and softened by inclusion of some actual descriptive data in the text.\n\nMention should be made that the high prevalence of some exposures means that the odds ratios are likely to be overestimates of actual risk.\n7. Discussion: This was the weakest part of the paper and could have been a lot stronger.\nThe section on bias again seemed rather ‘textbook’ in style and content, and does not convey the sense of a rigorous critical discussion of the specific issues raised by this study. More detail from the study itself and more detailed discussion of the individual points would help.\n\nThe authors raise as a limitation that the outcomes (presumably meaning back pain present or absent) are based on self-report – but it is difficult to see how the presence of back pain could be based on anything else but self-report. So this should rather have been a discussion of what the effects of misclassification of cases and controls might have been.\n\nThe sentence that states “it is anticipated that the current study’s prevalence underestimates the true burden of LBP in this study population” is decidedly odd, given the initial high response rate. This is where it would help to link the discussion back to the rationale for the study (what population is the prevalence trying to represent? How much bias could an initial 20% response possibly introduce?).\n\nThe final sentence of the discussion is not convincing.I don’t think it is right to say this is a “hypothesis generating” study – it does not fit with their main conclusion which urges all educational institutions in Kenya to look at how to modify risk factors in the workplace. This is an important conclusion but needs to be reached from (a) careful critical assessment of whether there study has established ‘cause’ or just association, and (b) rigorous comparison of their results with the wide international literature on these risk factors to see if their study is consistent with that literature and therefore strong enough to drive recommendations.\n\nFinally there should be a separate conclusion about the prevalence study, with suggestions of how the authors’ methodology might be used as the basis for more research to establish prevalence in other sectors of Kenyan society and why this might be useful to know. The authors are helpfully comprehensive about how their study fits with other prevalence studies in Africa – this is so important - but they could perhaps acknowledge that severity and impact must be added to future prevalence studies in their country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "70536", "date": "01 Oct 2020", "name": "Oyelola A. Adegboye", "expertise": [ "Reviewer Expertise Biostatistics", "exposure science", "multi-level modelling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMy comments majorly focused on the methods of this study:\nOne of the major weakness of this study is the use of “type of furniture” as a measure of sedentary behavior, this is not a sufficient risk factor. It is unclear to me why Q16 (hours of sitting) of the questionnaire was not actually explored together with the type of chair? The authors should at least acknowledge the lack of measurable factors for sedentary behavior, such as hours of sitting in their limitation. Additionally, there are several validated and standardized questionnaires for measuring some of the risk factors associated with LBP and sedentary, I am surprised that the authors did not explore this option. What is the reliability and validity of the test instrument used in this study?\n\nWhy was the study restricted to one college? In addition to teaching, I am assuming these staff also engaged in medical practices such as ward rounds and attending to patients, therefore it cannot be generalized to university teaching staff as the title suggests.\n\nRather than saying the significance of variables were evaluated at p≤0.2, they could have said “…predictors with p≤0.2 in the univariable analysis were included in the multivariable analysis.”\n\nThe authors sampled exactly 204 does not make sense, the authors should have invited an additional 10% participants to account for possible refusal as we have seen in this study, an actual sample size of 167 instead of the proposed 204 (80% power).\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\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-808
https://f1000research.com/articles/7-1738/v1	02 Nov 18	{ "type": "Case Report", "title": "Case Report: Resolution of chronic urticaria following treatment of odontogenic infection", "authors": [ "Susan Tadros", "Sameer Bahal", "Vasantha Nagendran", "Susan Tadros", "Vasantha Nagendran" ], "abstract": "Background: Chronic spontaneous urticaria (CSU) is a condition characterised by the presence of hives with/without angioedema, that affects individuals on more days than not for 6 weeks or more. The role of infection as a potential trigger for CSU is well described, but the current clinical guidelines do not recommend routine screening for underlying infections. Main observations: We report a case of severe prolonged chronic spontaneous urticaria in a 19-year-old, that went into rapid remission following the treatment of dental infection. Conclusions: Clinicians should recognise the potential role that infection can have in causing chronic urticaria. There should be a low threshold to treat infection in such circumstances.", "keywords": [ "Chronic Spontaneous Urticaria", "Dental Infection" ], "content": "Introduction\n\nUrticaria is a dermatological disorder that manifests as raised erythematous lesions that range in size. They are pruritic and typically resolve with no changes to the appearance of the skin. Urticarial lesions may be associated with episodes of swellings known as ‘angioedema’. The role of infection as a potential trigger for urticaria and angioedema is well described but the precise mechanism by which infection induces release of histamine from mast cells is unknown. Infections, including dental infections, have been associated with urticaria; however, current chronic urticaria guidelines do not recommend routine screening for underlying infection1. We report a case of severe chronic spontaneous urticaria that rapidly resolved following root canal treatment.\n\n\nCase report\n\nA 19-year-old male patient was referred to the Immunology clinic by his General Practitioner. He presented with a two-month history of urticaria with intermittent episodes of angioedema. His initial symptoms included facial pruritis, periorbital erythema and angioedema involving the upper and lower lips. Within 30 minutes of his first episode of angioedema, he developed widespread urticaria which responded to treatment with antihistamines. The following day, he experienced a recurrence of the symptoms and continued to have almost daily symptoms of urticaria with intermittent episodes of angioedema. He was commenced on an alternative anti-histamine by his GP but continued to develop urticaria and experience swellings of the hands and feet. His treatment was escalated at his initial visit to Immunology Clinic to fexofenadine 180mg twice a day with an additional 10–20mg of cetirizine. In addition, montelukast, a leukotriene receptor antagonist, was commenced.\n\nThe number of hives and degree of pruritis were graded using an objective scoring system known as the Urticaria Activity Score 7 (UAS7) that provides a weekly average score out of a maximum score of 40. The patient recorded weekly UAS7 scores of 30, despite treatment with maximum doses of antihistamines and montelukast. Therefore, Anti-IgE therapy with the monoclonal antibody ‘Omalizumab’ was offered. In the interim, he presented to his dentist with a broken tooth and was found to have carious molars requiring root canal treatment. One week after this intervention, his UAS7 score fell to 4 and then to 0, and he has remained in remission (UAS 7 score 0) for 9 months. As he was rather needle-phobic, he was delighted that this obviated the need for Omalizumab injections. Initial investigations including full blood count, renal function, liver function and thyroid function tests were all within the normal ranges.\n\n\nDiscussion\n\nChronic spontaneous urticaria (CSU) is defined as daily or almost daily urticaria for at least 6 weeks1. In up to 50% of patients, urticaria may be associated with episodes of angioedema2. These features are the result of degranulation of mast cells with the release of granule contents, predominantly histamine. Patients often present to their GP and are referred for further assessment and management by Immunologists, Allergists or Dermatologists when first line treatment with antihistamines fail to control the symptoms. The mainstay of treatment is high dose antihistamines and leukotriene receptor antagonists1. In recent years, the anti-IgE monoclonal antibody therapy, Omalizumab, has been used as an effective treatment for patients who fail to respond to first line therapy.\n\nIn cases of CSU, triggers such as food-based allergens or airborne allergens are rarely implicated3. In acute urticaria (defined as having a duration of less than 6 weeks), causes are more likely to be identified. In one study of 79 cases of acute urticaria, 36.7% were secondary to infection4.\n\nA number of studies have demonstrated an increased prevalence of oropharyngeal infections including dental infections, sinusitis and tonsillitis in patients with chronic urticaria2,5,6. An early study from 1964 demonstrated radiological evidence of focal dental infection in 29% of their cohort of patients with chronic urticaria2. In addition, cases have been reported of resolution of urticaria after treatment of dental infections7,8. In one case bacterial cultures from dental lesions grew the gram-negative bacteria Veillonella parvula9. It is thought that Lipopolysaccharide from gram negative bacteria induces an inflammatory response characterised by histamine release from mast cells and resulting urticaria10.\n\nThe presented case history demonstrates the close temporal relationship between treatment of dental infection and the improvement of urticaria and reduction in medication requirements. Inflammatory markers were not monitored in this case but may have been elevated11. Measurement of markers of the acute inflammatory response, including CRP, can easily be included in assessment of patients with chronic urticaria. Together with a careful history, an elevation in acute inflammatory markers, may highlight the presence of infection/inflammation. Where infection has been excluded, the elevated inflammatory markers may identify patients with more severe chronic urticaria12.\n\nOur patient had failed first and second line treatments for chronic urticaria with persistent and troublesome symptoms. With a UAS 7 >28, demonstrating poorly controlled chronic urticaria, he was eligible to commence anti-IgE therapy13. Monoclonal antibody anti-IgE treatment with Omalizumab is now provided by some immunology and dermatology units in the UK. Patients are given Omalizumab by sub-cutaneous injection once a month for 6 months, and their response is monitored throughout. Although relatively safe, any new treatment is not without the risk of side effects. In addition, the treatment is costly, and should be reserved for patients who have severe CSU that fail to respond to treatment with the maximum dose of anti-histamine treatment and leukotriene receptor antagonists.\n\nOur case history illustrates the importance of searching for infections, including odontogenic infections, prior to commencing immunosuppression or anti-IgE therapy in patients who are resistant to first line treatment of CSU.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of their clinical details.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nThe author(s) declared that no grant(s) were involved in supporting this work.\n\n\nReferences\n\nPowell RJ, Leech SC, Till S, et al.: BSACI guideline for the management of chronic urticaria and angioedema. Clin Exp Allergy. 2015; 45(3): 547–65. PubMed Abstract \| Publisher Full Text\n\nWedi B, Raap U, Wieczorek D, et al.: Urticaria and infections. Allergy Asthma Clin Immunol. 2009; 5(1): 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonroe EW: Urticaria. Int J Dermatol. 20(1): 32–40.\n\nKulthanan K, Chiawsirikajorn Y, Jiamton S: Acute urticaria: etiologies, clinical course and quality of life. Asian Pac J Allergy Immunol. 2008; 26(1): 1–9. PubMed Abstract\n\nBuss YA, Garrelfs UC, Sticherling M: Chronic urticaria--which clinical parameters are pathogenetically relevant? A retrospective investigation of 339 patients. J Dtsch Dermatol Ges. 2007; 5(1): 22–9. PubMed Abstract \| Publisher Full Text\n\nBrzewski PŁ, Spałkowska M, Podbielska M, et al.: The role of focal infections in the pathogenesis of psoriasis and chronic urticaria. Postepy Dermatol Alergol. 2013; 30(2): 77–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnger AH: Chronic urticaria. II. Association with dental infections. South Med J. 1960; 53: 178–81. PubMed Abstract\n\nTanphaichitr K: Chronic urticaria associated with bacterial infection. A case of dental infection. Cutis. 1981; 27(6): 653–6. PubMed Abstract\n\nSonoda T, Anan T, Ono K, et al.: Chronic urticaria associated with dental infection. Br J Dermatol. 2001; 145(3): 516–8. PubMed Abstract \| Publisher Full Text\n\nNygren H, Dahlén G: Complement-dependent histamine release from rat peritoneal mast cells, induced by lipopolysaccharides from Bacteroides oralis, Fusobacterium nucleatum and Veillonella parvula. J Oral Pathol. 1981; 10(2): 87–94. PubMed Abstract \| Publisher Full Text\n\nBansal T, Pandey A, D D, et al.: C-Reactive Protein (CRP) and its Association with Periodontal Disease: A Brief Review. J Clin Diagn Res. 2014; 8(7): ZE21–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKasperska-Zajac A, Grzanka A, Kowalczyk J, et al.: Refractory chronic spontaneous urticaria and permanent atrial fibrillation associated with dental infection: Mere coincidence or something more to it? Int J Immunopathol Pharmacol. 2016; 29(1): 112–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOmalizumab for previously treated chronic spontaneous urticaria \| Guidance and guidelines \| NICE [Internet]. [cited 2018 Jul 28]. Reference Source" }	[ { "id": "45040", "date": "01 Apr 2019", "name": "Sinisa Savic", "expertise": [ "Reviewer Expertise Clinician scientist (clinical immunology and allergy)", "ares of interest: auto inflammatory disorder", "chronic urticaria", "primary immunodeficiency", "drug allergies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report describes a patient who developed CSU refractory to 1st and 2nd line therapies. His CSU resolved promptly after treatment of the concurrent dental infection.\n\nThis case illustrates importance of searching for underlying causes of CSU as part of routine clinical assessment. The authors state that routine infection screen is not recommended by the current guidelines, however the guidelines state: ‘The diagnosis is based primarily on the clinical history. Investigations are determined by the clinical history and presentation, but may not be necessary”. Although infection screen is not specifically mentioned, taking a thorough clinical history as suggested by the guidelines should include enquiry about possible infections and subsequent investigations might include an infection screen.\n\nOne of the reasons why infections might be overlooked when assessing CSU, is that the past recommendations for routine screening for H. Pylori and its eradication failed to produce desired outcomes1. There are other examples in the literature where chronic infection has been linked with CSU, and one such example is HepC and HepB. However, a comprehensive review of the literature failed to find an obvious associations, and routine screening is not recommended2.\n\nIn summary, this is well written article with a useful message, but discussion regarding the role of infection triggers in CSU should be more comprehensive and include the points mentioned above.\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/7-1738
https://f1000research.com/articles/8-789/v1	05 Jun 19	{ "type": "Opinion Article", "title": "Better and fulfilling healthcare at lower costs: The need to manage health systems as complex adaptive systems", "authors": [ "Joachim P. Sturmberg", "Johannes Bircher", "Johannes Bircher" ], "abstract": "Rising healthcare costs are major concerns in most high-income countries. Yet, political measures to reduce costs have so far remained futile and have damaged the best interests of patients and citizen. We therefore explored the possibilities to analyze healthcare systems as a socially constructed complex adaptive system (CAS) and found that by their very nature such CAS tend not to respond as expected to top-down interventions. As CAS have emergent behaviors, the focus on their drivers – purpose, economy and behavioral norms – requires particular attention. First, the importance of understanding the purpose of health care as improvement of health and its experience has been emphasized by two recent complementary re-definitions of health and disease. The economic models underpinning today’s healthcare – profit maximization – have shifted the focus away from its main purpose. Second, although economic considerations are important, they must serve and not dominate the provision of healthcare delivery. Third, expected health professionals’ behavioral norms – to first consider the health and wellbeing of patients – have been codified in the universally accepted Declaration of Geneva 2017. Considering these three aspects it becomes clear that complex adaptive healthcare systems need mindful top-down/bottom-up leadership that supports the nature of innovation for health care driven by local needs. The systemic focus on improving people’s health will then result in significant cost reductions.", "keywords": [ "Healthcare costs", "Healthcare financing", "Healthcare as complex adaptive system", "Sense or purpose of healthcare", "Definition of health", "Healthcare organization", "Norms in healthcare", "Complex adaptive systems", "System dynamics", "Philosophy of medicine" ], "content": "Introduction\n\nIn high income countries healthcare* costs were rising more rapidly during the past decennia than gross domestic products, and this generally is considered not to be sustainable1. One important explanation for these observations is Baumol’s conclusion that growth of wages in excess of productivity growth drives growth of health care expenditure2. However, there is a poor correlation between health care system structures and spending with patient health outcomes (Table 1).\n\nThe table highlights the differences in health system performance amongst 4 selected OECD-countries with distinctively different health system structures. Performance outcomes arises from the unique dynamic behaviors of the system, i.e. outcomes cannot be attributed to one or two specific features of the system. It also means that direct comparison of outcomes between different systems is difficult as they depend on each system’s unique characteristics and dynamics.\n\nThe relative contributions of commonly intimated factors such as scientific and technological progress in medicine and changing age demographics on healthcare expenditure and/or health system performance remain uncertain.\n\nMajor efforts to lower healthcare expenditure by applying economic principles like fundholding, limiting services, capping or bundling payments, lean management, guidelines or pay-for-performance incentives have been tried in various jurisdictions; evaluations of these interventions on overall financial burden on society and/or patient/population health outcomes remain limited and unconvincing1. Economically driven initiatives demonstrably increased the administrative load of health care professionals as it has detracted them from their primary task – to expertly and professionally attend to patients care needs2,3. Studies have identified that newer technical equipment and newer drugs are two factors that unequivocally make health care more expensive4. In addition many physician activities and procedures are not truly purposeful for the achievement of better health, an observation that has led to the “Choosing Wisely”movement5. The relative contribution of this policy on healthcare costs and outcomes is outstanding.\n\nIn high income countries healthcare systems generally are organized top-down. This hierarchical structure goes from the health ministry all the way down to the youngest physicians, nurses and orderlies in hospitals or physician practices6. Since all coworkers must contribute according to rules from above, it is assumed that such systems lose an important part of their intrinsic motivation and productivity. Another method to organize health care would be bottom-up7,8. Such system organization implies that for each specific condition physicians and nurses who work with the patients know best how to optimally perform their work. Therefore, they are invited to first, continuously contribute to the system’s overall development and second, to adopt their own working rules, a feature that is applied to all scales of the organization (Figure 1). It has been hypothesized that bottom-up organizations create best adapted solutions to changing problems and needs, a hypothesis supported by the experiences of the NUKA health system in Alaska9,10 and the EDARP health system in Kenya11,12 and are detailed below.\n\nThe effects of the top-down policy-driven approach on health care delivery are illustrated by the ever-decreasing size of the inner circles from one organizational level to the next where each level further constrains what the next lower level can achieve – the top-down leadership’s constraints minimize bottom-up feedback (left). The bottom-up approach is illustrated by dotted circles – to emphasize the open and adaptive nature of entities at each level- all focused on the system’s overall goal. Every higher-level circle emerges as a result of various interactions (arrows) at a lower level, resulting in the variance of characteristics and behaviors that depend on unique local circumstances. While each level shows variability in its components, each level component is the best adapted version of this level in its unique environment, and each does uniquely contribute to the achievement of the overall policy goals & settings – leadership minimizes constraints and encourages constant feedback across all levels of the system (right). Note that the complexity of a system arises from the feedback loops between top-down and bottom-up interactions across all the layers of the system. These two approaches are not mutually exclusive, rather – as the figure highlights – reflect the tension in leadership between trust (minimize constraints, maximize contextual adaptation) and distrust (maximize constraints, minimize variability). For a detailed discussion on causation in complex adaptive systems see Ellis8; for a discussion on complex adaptive organizations see Laloux7.\n\nThis paper sets out to explore complex adaptive system (CAS) thinking to the organization and function of healthcare systems. Complex adaptive dynamics provide the theoretical basis to the structure and function of bottom-up organizations13. Initially we present the nature of a CAS as applied to healthcare including some possible perspectives for the improvement of healthcare systems in general. From this we will consider how CAS understandings may change healthcare systems and thereby benefit patients and healthcare personnel while simultaneously reducing costs.\n\n\nWhat is a CAS?\n\nIn general terms a CAS is an autonomously functioning open system separated from its surroundings by a fuzzy boundary, i.e. it can receive inputs from and provide outputs to its environment (Figure 2). Its inside is composed of active parts, called agents, that continuously and spontaneously interact with each other without external control. These interactions may be simple (i.e. linear and predictable) where cause and effect are fixed, complicated (still linear and predictable) where a particular cause results in a particular outcome (often with a delay in time or place), complex (i.e. nonlinear) where cause and effect are perceivable but not precisely predictable, or chaotic (i.e. unrelated) where no cause and effect relationship is evident. Interactions among the agents of a CAS result in feedback, and feedback drives the emergent behavior of the system as a whole13.\n\nComplex adaptive systems are open, i.e. they receive inputs from their external environment, and the interactions – especially feedback loop interactions – between its agents result in emergent outcomes that can be shared with external agents or other systems.\n\nTwo additional features contribute to the complex adaptive dynamics of a CAS. Firstly, many agents are CAS in their own right, i.e. they constitute subsystems, and vice versa, each CAS itself is part of a larger supra-system. This nested nature of CAS results in a hierarchical layering where higher layer supra-systems “constrain” the potential “bottom-up” emergent behavior of lower layer subsystems8. Secondly, the interdependencies between the nested hierarchical structure and the dynamics resulting from the opposing forces of “top-down” constraints and “bottom-up” emergent potentials makes CAS “stable and resilient” in constantly fluctuating environments (i.e. CAS are in a non-equilibrium state). Internal and external perturbations into a non-equilibrium system contribute to its emergence over time, and this may have no influence or enhance or diminish the system’s overall performance and stability. This means that a CAS can evolve in response to needs of its surroundings. Rarely is the input into a system large enough to cause a complete and/or abrupt system change.\n\n\nThe healthcare system is a “socially constructed” CAS\n\nHealthcare systems are “organizational systems”, thus they are socially constructed. An organizational CAS emerges based on purpose, goal and value propositions that give rise to its operating principles or driver. Combined they provide the “top-down” constraints that limit the “bottom-up” emergent possibilities of its agents at the various levels within the healthcare system (Figure 3).\n\nThe driver of the health system – resulting from its agreed purpose, goals and values – align and limit the potential interactions of its agents in response to diverse inputs. These constraints “determine” the potential outcomes the health system can deliver, both in terms of health outcomes for the patients treated and the economic and resource costs associated with the service delivery.\n\nBesides of health professionals and support workers a health system’s agents also include - amongst others - politicians, administrators, pharmaceutical organizations, devise makers and insurance companies. System “inputs” in the first instance consist of persons in need of better health. Other important inputs are resources like new knowledge, technologies, finances, drugs and technical equipment, etc. The overall performance of the CAS results in emergent “outputs”, i.e. “persons with improved health”.\n\nA healthcare system’s driver “focuses or directs” the activities of its agents. It tends to support influences that are consistent with its established purpose, goals and values. It thereby allows the emergence of appropriate structures and functions necessary for its overall performance. Thus, a health system’s driver may allow changes to the structure, e.g. the addition of a new health service division (structural change) or the implementation of a new service delivery approach (functional change). Success requires bottom-up adoption as the “current successful drivers” of a CAS tend to strongly resist top-down “instructions” that contradict, restrain or impede the status quo.\n\n\nThe role of governance - Top-down versus bottom-up\n\nA socially constructed CAS functions based on its socially constructed driver arising from the system’s definition of its purpose, goals and values statement. The driver ultimately can be “controlled” – in a governance sense – top-down “bureaucratic”, or bottom-up “grass-roots”.\n\nThe schematic comparison depicted in Table 2 reveals fundamental differences between these two types of governance. A traditional organizational system uses hierarchy and manages the organizations top-down. Motivation of coworkers is extrinsic, induced by command and control and human relationships are contractual. Superiors focus on the efficiency of the system and evaluate whether or not the activities are appropriate (process oriented). In contrast, governance in a complex adaptive organizational system is based on heterarchy and personal leaderships. The structure is bottom-up self-organizational. Motivation is intrinsic by identification with the purpose, goals and values of the organization. Human relationships are based on personal commitment and the focus of employees is problem-oriented. To supervise the organization the leadership assesses the outcome (outcomes oriented).\n\nThe principles of leadership between the two types of governance are fundamentally different. Leadership in hierarchical systems relies on power, command and control, whereas leadership in heterarchical system is based on collaboration, respect, learning from each other and measuring of outcomes7.\n\n\nFrom theory to first-hand experience\n\nObservation would suggest that it is always “easier” to live with the imperfection of the status quo and to fiddle with its imperfection at the margins – despite all the talk about the failing health systems around the world. The top-down improvement efforts of the past 30+ years have little to show for. However, there are some notable examples that support the hypothesis that bottom-up approaches create organizations that deliver highly adapted solutions to the changing problems and needs of their patients/communities in a more efficient and cost-effective way.\n\nThe Mayo brothers have been the first to organize their hospital-based health care around a system driver, codified in the mottoƗ of “The needs of the patient come first.”15.\n\nTo prevent monetary inducements influencing clinical decision-making the Mayo founders took the “radical step” to employ all physicians (and all other staff) on specialty adjusted fixed salaries. The hierarchy among physicians is flat, and accepted patients (based on a “medical needs assessment”) are treated irrespective of their capacity to pay. Importantly the clinic’s medical ethics are cultivated continuously and are self-reinforcing.\n\nFor many years the Mayo Clinic has now remained the number one healthcare organization for patient care in the United States. Careful consideration by its leadership of the sense or purpose of healthcare, its financing and physician ethics, i.e. the “driver” of the Mayo Clinic, have maintained its longstanding success in a constantly changing health care envirionment15. Today the Mayo Clinic is regarded as the best practice model of health service delivery in the US in a primarily tertiary oriented healthcare organization – achieving great health outcomes in a most cost-effective and efficient way.\n\nAn inadequate centrally controlled American Indian Health Service morphed into the highly functioning NUKA health system as a result of a bottom-up change to the system’s drivers. Alaskan native people realized a bottom-up customer owned system oriented toward physical, mental, emotional and spiritual wellness through community and interprofessional cooperation. The change of their health system’s driver to embrace “shared responsibility, commitment to quality and family wellness” achieved a healthcare service that “finally” meets its patients’ and community’s needs and aspirations. Ongoing collaboration ensures that the system remains responsive to the community’s evolving requirements as well as medical progress, something that the previous top-down organization by a Washington-based government bureaucracy could not achieve9,10.\n\nAs health systems are constructed socially “finding the right driver” – as illustrated by the Alaskan native people’s approach – can lead to the emergence of a health system that appropriately meets its users’ needs. When bottom-up “improvement of performance” is allowed to drive a healthcare system it can evolve to meet the system’s overarching goals and purposes while locally delivering amongst others patient centered care based on scientific progress and technological advances.\n\nBesides of being aligned with their patients’ needs and having achieved better health outcomes, the NUKA health system approach has also demonstrated that it has achieved these outcomes at lower costs10.\n\nEastern Deanery AIDS Relief Program (EDARP). The Eastern Deanery AIDS Relief Program (EDARP) is an example that demonstrates how a “clearly defined” driver can create a community-based health service “de-novo”. Initially the program solely aimed to relieve the suffering of dying AIDS patients. However, the community health workers involved in the care of these patients identified many additional interconnected needs – at the personal, social and community levels – that resulted in the emergence of a community led, community delivered health and social service network for a Nairobi slum district that has dramatically improved health outcomes at the personal and community levels for this disadvantaged population11,12.\n\nBuurtzorg – Dutch for “neighborhood care”. A fascinating example of bottom up governance has been realized in the Netherlands by the project “Buurtzorg‡”17. This is a pioneering organization established in 2006: A nurse-led bottom-up model of holistic care assumes responsibility for ambulatory nursing in the communities. It not only revolutionized community care, but client satisfaction rates are the highest of any health care organization; staff commitment and contentedness is superior. Ernst & Young documented savings to the Dutch health care system of around 40%, if all care would be provided this way18.\n\nA care system for Indigenous people in Odisha-India. Another example showing a bottom-up success concerns health services in villages of recently settled indigenous people in Odisha, India. These villages received top down basic health care by the Indian government. Yet, many villagers refused vaccinations of their children and used supplied mosquito nets just for fishing. When members of an NGO that had cared for the development of these villages explained the Meikirch model of health to the inhabitants their health-related behavior improved markedly. Ninety percent of informed villagers washed their hands before meals, while in control villages without teaching only 41% did it. Eighty percent of households had latrines in comparison to 42% in control villages, and 98% of children were vaccinated compared to 58% in control villages. These results confirm that indigenous villagers do not respond satisfactorily to “gifts” from the government, but they can understand teaching about health and correspondingly change their behavior19.\n\n\nWhat has been learnt\n\nA complex adaptive organization is in constant flux responding to diverse external inputs that challenge its internal structures and dynamics. Lived purpose, goals and values statements are the basis for a system’s driver that ultimately governs the behavior of complex adaptive organizations and ensures a level of dynamic stability. Prevailing top-down organizational leadership, based on command, power and control, invariably results in limited emergent staff engagement stifling staff morale, and in turn diminishes their creativity and productivity. Alternatively, organizations can adopt a bottom-up management approach fostering collaboration, respect and learning, making the organization more resilient.\n\nBottom-up minded complex adaptive organizations usually have a well-defined purpose, clearly discernable goals and transparent values that together give rise to the system’s driver. Three features underpin an effective driver of a bottom-up health system or service: a focus on health, minimizing financial collusion, and adhering to Hippocratic norms of the medical professions13.\n\nThe difference between these two leadership mindsets is the nature of the constraints created – the more restrictive they are the more they limited what staff at each organizational level can do. Neither leadership style changes the fact that leaders are ultimately responsible for their organization’s performance and achievements.\n\nRestoring or improving a person’s health is the core purpose of health care delivery. Hence, “improvement of health” must be part of the driver for health care, although “improved health” until now could only be understood intuitively and has only tacitly shaped patient/physician interactions.\n\nThe lack of a precise definition of health, and as a corollary disease, has remained an important defect. Without a clear understanding of the meaning of health healthcare systems cannot offer an enabling vision to its staff and their patients, and unsurprisingly allowed arbitrariness in decision-making and management based on economic or personal interests. Over the last 5 years, science based models that define health and disease have emerged – the first being the Meikirch-model20,21 whose fundamental tenet has recently been corroborated by a multi-disciplinary group collaboration demonstrating the interconnectedness and interdependence of external and internal variables on the dynamic state of health22. The purpose of healthcare therefore no longer remains intuitive and difficult to communicate. It can now be analyzed and expressed explicitly. As a result, it is possible to devote the attention of physicians, nurses and other health care workers to each individual patient’s existential health needs. Today we are able to “scientifically” reconnect with our predecessors who devoted their lives as nurses or physicians to this fulfilling task with financial modesty and much personal satisfaction.\n\nIn recent decades health professionals’ attitudes and approaches to health care delivery have become compromised by focusing on profit maximization. As a result, financial interests have distorted health care away from its central mission: Patients are no longer certain that they are advised only according to their personal health needs rather than being seen as the means to achieve the financial interests of institutions or some of their health professionals.\n\nToday health professionals struggle with the tensions arising from their core duty of meeting the health needs of their patients and the pressures exerted on them to practice within the limited financial resources provided to them. While there clearly is a limit to resourcing health care systems one nevertheless has to acknowledge that almost all medical decision-making has a wide margin of discretion. Organizational leaders easily (and frequently) modified discretional decision-making applying external forces, like financial incentives, nudging and competition23,24.\n\nUnquestionably though, financing of a complex adaptive healthcare organization should have no other purpose than to provide adequate resources to deliver needed health care services to its patients/communities. Unfortunately, in the past decades financial pressures have been applied increasingly and widely. They have been used to increase physicians’ “productivity”, to modify their behaviors in relation to diagnostic and therapeutic approaches as well as for the specific purpose to reduce overall healthcare costs.\n\nYet, financially driven interventions tend to disregard the best interest of patients, and have failed to diminish costs, but – as an unintended consequence – have resulted in delayed access to healthcare and increased costs25. In most cases financial incentives only temporarily changed incentivized clinician behaviors but more importantly they damaged health system design and/or health outcomes26,27.\n\nIn addition, excessive advertising of technologies to attract patients to specific institutions also have influenced and deteriorated patient care and increased waste28,29. Simultaneously excessive administration and prolonged working hours proved harmful for health service personnel29,30. Although originally intended to reduce healthcare costs, top-down politically or economically motivated measures have augmented administrative workloads and resulted in increasing frustration followed by an exodus of physicians and nurses29.\n\nThe sum of these observations confirms that first priority cost-containment as a driver of a healthcare system by necessity leads to failure. Health system financing is important, but it must serve medical care delivery that improves patients’ health. It must not direct it. That said, there equally is no place for waste in health care – whilst highest quality at the lowest possible cost remains a priority, improvement of a patient’s health must be respected as the first priority.\n\nSince antiquity ethical norms have played important roles in all spheres of life. Confucius’ golden rule is a most famous example: “Never impose on others what you would not choose for yourself.” He lived in China from 551 to 479 AC. Around the same time, at the other side of the world, the Hippocratic oath emerged as the guiding frame for the conduct of physicians. Since 1948 it has been periodically updated by the World Medical Association and renamed “Declaration of Geneva”. The most recent revision in October 201731 begins with this affirmation: “As a member of the medical profession I solemnly pledge to dedicate my life to the service of humanity. The health and wellbeing of my patient will be my first consideration.” Importantly, the declaration does not concern itself with the income of physicians, however, by implication may allude to a physician’s financial responsibilities in a later statement: “I will practice my profession with conscience and dignity and in accordance with good medical practice.” Of note, the Geneva Declaration expresses fidelity toward patients and the physician’s personal integrity while tacitly acknowledging economic and financial concerns.\n\n\nConclusions: Respecting the nature of complex adaptive health systems achieves better health outcomes at lower cost\n\nHealthcare systems are socially constructed complex adaptive organizations. As other complex adaptive systems they are driven by three components, their explicitly expressed purpose, their goals and their values. First examples of CAS are the Buurtzorg ambulatory nursing, the NUKA or the EDARP health systems. These organizations fulfill their purpose by having created “loose enough constraints” that foster bottom-up emergent behaviors enabling their staff to adaptively respond to changing patient needs and economic constraints.\n\nThese deliberations and examples demonstrate that it is indeed possible to develop and adjust the driver of a CAS in the combined best interest of patients and society. Successful complex adaptive organizations have distributed leadership that fosters collaborative learning to adapt to the changing needs of its patients. For the society they promise to be more effective and more efficient at reduced costs.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this study.\n\n\nFootnotes\n\n*Healthcare refers to the institution, health care to the deliverables of its agents\n\nƗAn interesting example of attention to the driver of healthcare is given by the Mayo Clinic in Rochester Minnesota, USA. In 1910, William James Mayo, M.D., delivered the commencement address at Rush Medical College in Chicago declaring that: \"The best interest of the patient is the only interest to be considered, and in order that the sick may have the benefit of advancing knowledge, a union of forces is necessary.\" This ethos ever since has driven the Clinic’s approach to patient care.\n\n‡translates as “neighborhood care”\n\n\nReferences\n\nWHO: WHO New Perspectives on Global Health Spending for Universal Health Coverage European Region Health Expenditure Dashboard, 2000–2015. In: Global health spedning. Reference Source\n\nHartwig J, Sturm JE: Robust determinants of health care expenditure growth. Appl Econ. 2014; 46(36): 4455–74. Publisher Full Text\n\nFaller MS, Gates MG, Georges JM, et al.: Work-related burnout, job satisfaction, intent to leave, and nurse-assessed quality of care among travel nurses. J Nurs Adm. 2011; 41(2): 71–7. PubMed Abstract \| Publisher Full Text\n\nBodenheimer T: High and rising health care costs. Part 2: technologic innovation. Ann Intern Med. 2005; 142(11): 932–7. PubMed Abstract \| Publisher Full Text\n\nLehmann C, Berner R, Bogner JR, et al.: The \"Choosing Wisely\" initiative in infectious diseases. Infection. 2017; 45(3): 263–8. PubMed Abstract \| Publisher Full Text\n\nUvhagen H, Hasson H, Hansson J, et al.: Leading top-down implementation processes: a qualitative study on the role of managers. BMC Health Serv Res. 2018; 18(1): 562. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaloux F: Reinventing Organizations, A Guide to Creating Organizations Inspired by the Next Stage of Human Consciousness. Knowledge Partners, Maharashtra India, 2018.\n\nEllis GF: Top-down causation and emergence: some comments on mechanisms. Interface Focus. 2012; 2(1): 126–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGottlieb K: The Nuka System of Care: improving health through ownership and relationships. Int J Circumpolar Heal. 2013; 72(1): 21118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollinss B: Intentional whole health system redesign: Southcentral Foundation’s ‘Nuka’ system of care. Kings Fund. 2015. Reference Source\n\nNjoroge A, Cassidy S, Williams V: Making patient-centred care a reality in the slums of eastern Nairobi. Int J Tuberc Lung Dis. 2013; 17(10 Suppl 1): 5–8. PubMed Abstract \| Publisher Full Text\n\nSturmberg JP, Njoroge A: People-centred health systems, a bottom-up approach: where theory meets empery. J Eval Clin Pract. 2017; 23(2): 467–73. PubMed Abstract \| Publisher Full Text\n\nSturmberg JP: Health System Redesign. 2018. Publisher Full Text\n\nOECD: Health at a Glance 2017: OECD Indicators. OECD Publishing, Paris, 2017. Publisher Full Text\n\nBerry L, Seltman K: Management Lessons from Mayo Clinic: Inside One of the World’s Most Admired Service Organizations. McGraw-Hill, New York, 2008. Reference Source\n\nRouse WB: Health Care as a Complex Adaptive System: Implications for Design and Management. Bridg. 2008; 38: 17–25. Reference Source\n\nMonsen KA, de Blok J: Buurtzorg: Nurse-Led Community Care. Creat Nurs. 2018; 24(1): 112–7. PubMed Abstract \| Publisher Full Text\n\nBuurtzorg. Reference Source\n\nSamal S, Mohanti D, Born E, et al.: Teaching of health with the Meikirch model to indigenous people improves their health-supporting behavior: A pilot study. Med J DY Patil Univ. 2017; 10(1): 17–20. Publisher Full Text\n\nBircher J, Kuruvilla S: Defining health by addressing individual, social, and environmental determinants: new opportunities for health care and public health. J Public Health Policy. 2014; 35(3): 363–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBircher J, Hahn EG: Understanding the nature of health: New perspectives for medicine and public health. Improved wellbeing at lower costs [version 1; peer review: 2 approved]. F1000Res. 2016; 5: 167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSturmberg JP, Picard M, Aron DC, et al.: Health and Disease-Emergent States Resulting From Adaptive Social and Biological Network Interactions. Front Med (Lausanne). 2019; 6: 59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLagarde M, Blaauw D: Physicians' responses to financial and social incentives: A medically framed real effort experiment. Soc Sci Med. 2017; 179: 147–59. PubMed Abstract \| Publisher Full Text\n\nDoran T, Maurer KA, Ryan AM: Impact of Provider Incentives on Quality and Value of Health Care. Annu Rev Public Health. 2017; 38: 449–65. PubMed Abstract \| Publisher Full Text\n\nHaren MC, McConnell K, Shinn AF: Increased Patient Cost-Sharing, Weak US Economy, and Poor Health Habits: Implications for Employers and Insurers. Am Heal Drug Benefits. 2009; 2(3): 134–41. PubMed Abstract \| Free Full Text\n\nAlshamsan R, Majeed A, Ashworth M: Impact of pay for performance on inequalities in health care: systematic review. J Heal Serv Res Policy. 2010; 15(3): 178–184. PubMed Abstract \| Publisher Full Text\n\nRyan AM, Krinsky S, Kontopantelis E, et al.: Long-term evidence for the effect of pay-for-performance in primary care on mortality in the UK: a population study. Lancet. 2016; 388(10041): 268–74. PubMed Abstract \| Publisher Full Text\n\nBerwick DM, Hackbarth AD: Eliminating waste in US health care. JAMA. 2012; 307(14): 1513–6. PubMed Abstract \| Publisher Full Text\n\nAlexander D: How Do Doctors Respond to Incentives? Unintended Consequences of Paying Doctors to Reduce Costs. Work Pap Ser WP-2017–9, Fed Reserv Bank Chicago. 2017. Reference Source\n\nWehkamp KH, Heinz N: Ökonomisierung patientenbezogener Entscheidungen im Krankenhaus. Dtsch Arztebl. 2017; 114(47): 797–804. Publisher Full Text\n\nWorld Medical Association. Declaration of Geneva. 2017. Reference Source" }	[ { "id": "52863", "date": "27 Aug 2019", "name": "Jochen Hartwig", "expertise": [ "Reviewer Expertise Economics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors argue in favor of organizing healthcare systems bottom-up instead of top-down and discuss several examples of successful implementation of that principle around the world.\nThe contribution is an ‘opinion article’. Whether or not I agree with the authors´ opinion is not relevant. In my view, the article is a stimulating contribution to the debate on healthcare systems reform and should be indexed as such.\nFor a final version of the paper I suggest explaining \"the Meikirch model\" on p. 7 where it is first mentioned. In the fourth paragraph on the left hand side of p. 8 I think it should read 'limit' instead of 'limited'. Finally, I recommend dropping the first sentence on the right hand side of p. 8 because, in my view, it sounds a bit too lofty for a scholarly article.\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": "57136", "date": "06 Dec 2019", "name": "Wikum Jayatunga", "expertise": [ "Reviewer Expertise Public Health", "Health Economics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written opinion article of a topic of significant topical relevance as many developed health systems grapple with the challenges of growing healthcare needs with finite resources. Increasingly complexity science is being used to understand the behaviour and performance of healthcare systems and their agents, and this paper makes a convincing case for the importance of values/purpose/goals (which are sometimes neglected over financial/economic concerns).\nThe logic and flow of the argument is sound and well-supported by the cited literature. While the benefits of bottom-up approaches to leadership are the main thrust of the paper, with interesting case studies used as examples, the paper is measured in its conclusions: recognising that the top-down and bottom-up approaches are not mutually exclusive.\nI suggest revision of the following sentence in the section 'From theory to first-hand experience'. \"Observation would suggest that it is always “easier” to live with the imperfection of the status quo and to fiddle with its imperfection at the margins.\" As the writing style feels out of place, although this is only a minor point.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-789
https://f1000research.com/articles/7-1955/v1	19 Dec 18	{ "type": "Software Tool Article", "title": "Carrying out streamlined routine data analyses with reports for observational studies: introduction to a series of generic SAS® macros", "authors": [ "Yuan Liu", "Dana C. Nickleach", "Chao Zhang", "Jeffrey M. Switchenko", "Jeanne Kowalski", "Dana C. Nickleach", "Chao Zhang", "Jeffrey M. Switchenko", "Jeanne Kowalski" ], "abstract": "For a typical medical research project based on observational data, sequential routine analyses are often essential to comprehend the data on hand and to draw valid conclusions. However, generating reports in SAS® for routine analyses can be a time-consuming and tedious process, especially when dealing with large databases with a massive number of variables in an iterative and collaborative research environment. In this work, we present a general workflow of research based on an observational database and a series of SAS® macros that fits this framework, which covers a streamlined data analyses and produces journal-quality summary tables. The system is generic enough to fit a variety of research projects and enables researchers to build a highly organized and concise coding for quick updates as research evolves. The result reports promote communication in collaborations and will escort the research with ease and efficiency.", "keywords": [ "SAS® macros", "observational studies", "streamlined data process", "reporting", "collaborative", "Good-Research-Practice" ], "content": "Introduction\n\nThe increasing availability of large-scale medical registry databases (e.g. SEER1, NCDB2), health insurance claim databases (e.g. the US Food and Drug Administration’s Sentinel Initiative3, MarketScan Research Database4), electronic medical record databases (EMR), or secondary data from clinical trials provides opportunities for researchers and policymakers to address a variety of clinical practice questions and make informed decisions. A retrospective or observational study based on such data allows researchers to examine medical care in a real-life setting, and, if carefully done, generalize results to an extended population and clinical setting. With a large pool of patients, longer follow-up periods, and an affordable cost, such studies can address broader research questions with deeper insights. Such study designs also hold inherent limitations, such as selection bias (e.g., certain groups of patients are more likely to access a certain therapy) and confounding (e.g., the observed treatment effect might mix with the effects of other important prognostics factors that are imbalanced among treatment arms). It is believed that through thoughtful design, careful analysis, accurate interpretation, and transparent reporting, a sound scientific conclusion can be reached with minimized limitations5–7. However, even when well equipped with the concepts of good research practice8–12, a researcher holding a promising hypothesis with access to an excellent data source may face many challenges. They may include a lack of understanding of the full extent of the massive data and its feasibility to answer the study question(s); the complexity of the data on hand; the need for tediously repetitive and time-consuming data processing; lack of transparency in data processing and reporting; or miscommunication among collaborators with mixed levels of experience and expertise. The main motivation of this work is to illustrate generic research and analytic framework for studies based on an observational database, to emphasize the importance of routine data analysis, and to introduce a series of SAS® macros13 designed to aid the journey of research with ease and efficiency.\n\nIn the following section, we illustrate how the proposed SAS® macros fit into the research and analytic framework seamlessly and assist in improving the overall research quality. In the case study, we exemplify the usage of the proposed macros by a real-life research project based on the NCDB with detailed result interpretations and discussion.\n\n\nMethods\n\nA general process of conducting research based on observational/retrospective studies involves a few general steps: study design, data management, data analyses, and reporting/review (shown in Figure 1), and each step interacts with each other as research is refined over time.\n\n1. At the study design phase, the primary study goals or hypotheses need to be stated clearly with a suitable database along with proper definitions of study population, outcomes, cohorts, and covariates. A comprehensive literature review in the area further assists a better study design.\n\n2. The data management step includes crafting the target study population by applying exclusion/inclusion criteria and preparing variables, such as creating derived variables, categorizing continuous variables, collapsing levels in categorical variables, handling missing values and outliers, etc. We recommend to assign an interpretable label and format to each variable for the best readable output table from the macros.\n\n3. In the data analyses step, different layers of information about the data will be unfolded by sequential analytic steps to test ultimate study hypotheses. The routine data analyses, followed by more advanced analytic approaches, allow for building a pyramid of evidence to support hypotheses and hence strengthen study conclusions.\n\n4. At the phase of reporting/review, we wrap up all results with interpretations in the context of the scientific background to evaluate the findings and draw conclusions with a statement of limitations. The process may involve reviews from an internal collaborative group or criticisms from journal reviews. There is some helpful guidance out there for reporting results from an observational study7,11.\n\nThe research itself should be dynamic and iterative as illustrated in Figure 1. Any issues, questions, criticisms, or new ideas that arise at a given step will redirect us to the previous steps and may lead to modifications of or additions to the original study design. The proposed SAS® macros will fit into this research framework seamlessly by covering routine data analyses in Step 3 more efficiently and enhancing reporting and communication in Step 4.\n\nThe routine data analyses serve as the foundational knowledge to support final claims in the research. The lack of correct perception or understanding of the importance of routine data analyses can lead researchers to rush into the final results without seeing the holes in the foundation, losing opportunities for improvement, and drawing biased conclusions. 1) For descriptive analyses, we get a chance to review the landscape of the entire study population and know the boundaries that apply to the conclusion. For example, if the study population is 99.9% Caucasian and 0.1% African American, the research findings may not be generalizable to the African American population or other racial groups. In addition, descriptive analyses help us to assess missing data, outliers, or possible data entry errors; and lead to additional data management. 2) For univariate associations or modeling, a natural association without any adjustment in the relationship between study cohorts and covariates or between outcome and cohorts/covariates will be demonstrated. We will learn of differences in patients’ characteristics among comparison cohorts, which may explain the differences we observe in the associations with the outcomes, especially when those characteristics are strong predictors of the outcomes. The phenomenon is referred to as selection bias or confounding, as commonly seen in observational studies. A confounder can be identified conceptually through existing knowledge or by examining the univariate associations as described here. 3) Both multivariable analyses and subgroup analyses are ways to deal with the confounding effects. Multivariable analyses are also known as main effects models which estimate an adjusted association with the outcome for each variable assuming all other variables are held constant in the model. The main effects model assumes the adjusted association with the outcome for each variable is uniformly constant across the levels of all other variables in the model. When such an assumption does not hold, subgroup analyses can be applied, known as interaction models, by which the association with the outcome is allowed to vary by the levels of a third variable (effect modifiers). Deeper technique details and strategies for multivariable modeling and variable selection, or more advanced approaches, such as propensity score methods, can be found in statistical textbooks or literatures14,15.\n\nSAS® macros are powerful tools and have been used widely to build customized SAS® procedures to reduce repetitive coding. Many individual SAS® macros were created for reporting purposes, but only suit very specific scenarios, and none of them cover a streamlined data analyses to truly serve the research generically and dynamically, as the proposed macros do.\n\nThe basic mechanism is similar in all proposed macros:\n\n1. Process each variable in the list of interest or each analytic step one at a time, and continue such iteration until the last variable or step.\n\n2. Inside each iteration, related SAS® exsiting procedures are carried out and the relevant and key information from the output is exported into SAS® datasets using ODS OUTPUT option.\n\n3. Organize all information collected in step 2 into a concise and interpretable format by a series of DATA steps.\n\n4. Report the final summary tables into a rich text file using PROC REPORT and ODS RTF option.\n\nAll proposed SAS® macros work under SAS 9.3 and above (English version).\n\nIn Box 1, we list the proposed SAS® macros and their descriptions. They were developed in a highly collaborative and fast-paced National Cancer Institute-Designated Comprehensive Cancer Center research facility to help manage the standard workload involved in multiple, contemporaneous studies. They can handle a large number of variables at one time and create professional, consistent, and interpretable summary tables, which often can be inserted into the manuscript directly with minor editing. Also, using those macros will result in readable and concise SAS code, and enable easier maintenance and revisions as the project evolves.\n\n\nUse case\n\nThe traditional surgical approach for early-stage lung cancer is via a thoracotomy, and such open chest surgery is limited to certain eligible patients due to the increased risk of mortality, especially among elderly patients with multiple comorbidities. Over the past two decades, video-assisted thoracic surgery (VATS) has been increasingly used in clinics and provides excellent short-term advantages over thoracotomy, such as fewer complications, less pain, improved lung function, shorter recovery period and lower costs. On the other hand, incomplete lymph node evaluation with VATS or a high rate of the residual tumor may compromise the long-term efficacy of VATS vs. thoracotomy (open). The knowledge uncertainty still exists regarding the optimal surgical approach for early-stage lung cancer concerning long-term survival16. The goal of this study is to compare the overall survival (OS) between two surgical approaches among the target eligible lung cancer patients.\n\nThe National Cancer Database (NCDB)2, a joint project of the American Cancer Society and the Commission on Cancer of the American College of Surgeons, was queried for non-small cell lung cancer patients with clinical stage < T2N0M0 who underwent lobectomy in 2010-2013. After applying the exclusion/inclusion criteria, we identified 34,048 eligible patients. The study cohort is the surgical approach (open vs. minimally invasive). The clinical outcomes of interest were 30-day mortality after surgery (yes vs. no) and overall survival (OS): defined as months from the date of surgery to death or last follow up. Covariates considered included sex, Charlson-Deyo score (comorbidity), year of diagnosis, histology, AJCC clinic T stage, age at diagnosis and urban/rural 2003. For a detailed definition of these variables, users can refer to the online data dictionary (http://ncdbpuf.facs.org/node/259?q=print-pdf-all).\n\nIn this case study, we show sequential analytical steps along with interpretations to probe into the study question by using the proposed SAS® macros. To make it easy to follow, we only include a short but relevant list of variables and cover routine data analyses. A comprehensive and complete manuscript for the study question is under preparation for a peer-reviewed publication.\n\nPlease note that these macros do not handle data cleaning or management directly. For the most interpretable results, it is highly recommended to properly format and label each variable before running these macros. All output tables are in Rich Text Format (RTF) and editable in Word.\n\nDescriptive Statistics\n\nThe macro %DESCRIPTIVE was used to generate Table 1. The table provides an overview of the study population’s basic characteristics, in which the frequency and proportion are listed for categorical covariates and summary statistics (mean, median, min, max, standard deviation) for numerical covariates, along with the frequency of missing values. In the following SAS® code example, a few universal macro variables are implemented: DATASET for the data set name; CLIST for a list of categorical covariates separated by space; NLIST for a list of continuous covariates separated by spaces; OUTPATH for the file path to store the output RTF file; FNAME for the name of the RTF file; DEBUG to suppress deleting intermediate data sets for error checking by setting to T. Setting the DICTIONARY option to T creates two additional columns in the summary table, for the covariates’ SAS® names and unformatted values. These columns are useful for the programmer to connect the table with the dataset and code. In Table 1, we observe that 35.6% of the study population underwent a minimally invasive surgical approach, 45% were male, 80.9% resided in a metro area, and 85.4% had comorbidity score ≤1.\n\n********** SAS® code example for %DESCRIPTIVE ***********************;\n\n/ TIP 1: Create the macro variable &DIR to specify the location used to store all results. If you are conducting an updated analysis, you need to change this path, and all updated results will be saved to a new location without modifying it in every macro. /\n\n%let dir = C:\\Desired Location to Save All Results\\;\n\n/ TIP 2: Create the macro variables &cat_var and &num_var to store categorical and numerical variable lists outside macros, and reference them in all related macros. All related tables can be updated by changing those two macro variables.\n\n/* TIP 3: Also note that the order of variables in CLIST is the same as the order in which they will appear in the final output table. Listing similar types of variables together, e.g., demographic variables, tumor characteristic variables, lab values, etc., will improve the readability of the results. /\n\n/ Variable Label:\n\nSEX - Sex\n\nUR_CD_03 -- Urban/Rural 2003\n\nCDCC_TOTAL -- Charlson Deyo score (comorbidity)\n\nYEAR_OF_DIAGNOSIS -- Year of Diagnosis\n\nHIS_CAT -- Histology\n\nTNM_CLIN_T -- AJCC Clinical T stage\n\nSURG_APP -- Surgical Approach\n\n/\n\n%let cat_var = sex UR_CD_03 CDCC_TOTAL YEAR_OF_DIAGNOSIS HIS_CAT TNM_CLIN_T;\n\n%let num_var = age ;\n\nTITLE 'Table 1 Descriptive Statistics for All Variables';\n\n%DESCRIPTIVE(DATASET=anal,\n\nCLIST = surg_app &cat_var,\n\nNLIST = &num_var,\n\nOUTPATH= &dir,\n\nFNAME=Table 1 Descriptive Statistics for All Variables,\n\nDEBUG=F);\n\nTITLE;\n\n************************************************************************;\n\nUnivariate Association with a categorical outcome/exposure variable.\n\nWe examined the univariate association between each covariate and surgical approach (Table 2) using %UNI_CAT and 30-day mortality after surgery (Table 3) using %UNI_LOGREG. Each covariate was processed separately, but summarized all together in one table. %UNI_CAT is suitable to compare multiple covariates specified in CLIST and NLIST between two or more cohorts defined by a categorical variable (OUTCOME) (see following SAS® code example). For each categorical covariate, frequencies from a contingency table are reported along with row percentages (Row%) or column percentages (Col%), which can be controlled by the ROWPERCENT option based on the desired interpretation. For each numeric covariate, summary statistics will be generated for each level of OUTCOME. The univariate associations can be tested by either parametric or non-parametric tests using the NONPAR option. The name of the tests will appear in the footnote of the table. Analyses can be performed using logistic regression models if the outcome/exposure variable is binary or ordinal using %UNI_LOGREG. As shown in Table 3, the probability of the EVENT = ‘Yes’ was modeled and the odds ratio (OR) was reported with a 95% confidence interval (CI). The reference level of a categorical covariate in CLIST can be chosen to aid interpretation, which can be done by separating the CLIST variables by an asterisk (), and then adding “(DESC)” or “(ref = “Reference level in formatted value”)” after each desired variable name (see following code). If the outcome/explanatory variable is numeric, users can refer to SAS® macro %UNI_NUM. In all report tables, a p-value < 0.05 will be shown in bold for easy visualization.\n\nThe parametric p-value is calculated by ANOVA for numerical covariates and chi-square test for categorical covariates.\n\nIn Table 2, 42.8% of patients that had minimally invasive surgery were male, and 46.2% among patients that had open surgery. Compared to open surgical patients, minimally invasive surgical patients were more likely to reside in metro areas (84.2% vs. 79.1%), to be diagnosed in recent years, to have a histology of adenocarcinomas (64.5% vs. 60.2%), and to be at clinical T stage 1 (70.5% vs. 64.5%) (all p < 0.001). In Table 3, the factors associated with a higher risk of 30-day mortality after surgery were having open surgery (OR = 1.48), male gender (OR = 2.16), older age (OR = 1.06 for every 1-year increase), a comorbidity score larger than one (OR = 1.8 compared to score = 0), squamous cell carcinomas (OR = 2.49 compared to adenocarcinomas), and clinical T stage 2 (OR = 1.34) (all p < 0.001). By interpreting the information in Table 2 and Table 3 together, the univariate association between surgical type and 30-day mortality could be confounded by the effect from histology and clinical T stage. Open surgery was more likely to be conducted among patients who had squamous cell carcinomas and clinical T2, and these two factors were linked to a higher rate of 30-day mortality. The observed higher probability of 30-day mortality in open surgery patients might be partially due to the imbalanced distribution of histology and clinical T stage in the surgical cohorts. The common statistical approaches to control for confounding effects include multivariable analysis, subgroup analysis, and propensity score methods.\n\n******* SAS® code example for %UNI_CAT and %UNI_LOGREG **********;\n\n/ TIP 4: In the following example, the NONPAR option is turned off because it will take more time to compute Fisher’s exact test on a large sample. /\n\nTITLE 'Table 2 Univariate Association with Study Cohort';\n\n%UNI_CAT (\n\nDATASET = anal,\n\nOUTCOME = surg_app,\n\nCLIST = &cat_var,\n\nNLIST = &num_var,\n\nNONPAR = F,\n\nROWPERCENT = F,\n\nSPREAD = T,\n\nOUTPATH = &dir,\n\nFNAME =Table 2 Univariate Association with Study Cohort);\n\n/ TIP 5: If you need to specify the reference level of a categorical variable, separate CLIST by “” and add (DESC) or (ref = “Reference level in formatted value”) after each desired variable name. Otherwise, CLIST can be separated by spaces alone. /\n\n%let cat_var_ref = SEX * UR_CD_03(DESC) * CDCC_TOTAL(DESC) * YEAR_OF_DIAGNOSIS * HIS_CAT(ref=\"Adenocarcinomas\") * TNM_CLIN_T(DESC);\n\nTITLE 'Table 3 Univariate Association with 30-day Mortality';\n\n%UNI_LOGREG(\n\nDATASET = anal,\n\nOUTCOME = PUF_30_DAY_MORT_CD,\n\nEVENT = 'Yes',\n\nCLIST = surg_app * &cat_var_ref,\n\nNLIST = &num_var,\n\nOUTPATH = &dir,\n\nFNAME = Table 3 Univariate Association with 30-day Mortality);\n\nTITLE;\n\n**************************************************************************;\n\nUnivariate Association with a time-to-event outcome\n\nWe examined the association of the study cohorts and each covariate with OS using %UNI_PHREG, as shown in Table 4. Each covariate was individually fit in a proportional hazards model (PROC PHREG in SAS®), and the hazard ratios (95% CI) and p-values were summarized in one table. In the following SAS® code example, the survival time variable is specified in EVENT, and the censoring indicator variable in CENSOR. Note that it requires that ‘1’ is used for the event and ‘0’ for the censored cases in the data (DATASET). Other options include LOGRANK to output log-rank p-value, TYPE3 to output the Type3 p-values for categorical covariates, and PHA for proportional hazard assumption checks. Similar to %UNI_LOGREG, one can set the reference level of a categorical covariate in CLIST. %UNI_PHREG can also handle time to event data in the counting process data form by using the START and STOP options, especially when modeling data with time-varying covariates or recurrent event data. It can also handle competing risk data by using the EVENTCODE option to activate the Fine and Gray’s model17.\n\nIn the univariate analysis shown in Table 4, we see that patients who underwent open surgery had 18% more chance to die before those who underwent minimally invasive surgery (HR = 1.18; 95%CI = (1.13-1.24); p < 0.001). Also, residing in rural or urban areas, higher comorbidity scores, squamous cell carcinomas, clinical stage T2, and older age were all risk factors linked to worse overall survival in this study population. In combination with the results in Table 2, those prognostic factors except for age were also more likely to present in patients who underwent open surgery and should be controlled for as confounders in multivariable analyses.\n\n******** SAS® code example for %UNI_PHREG and its options **************;\n\nTITLE 'Table 4 Univariate association with Overall Survival';\n\n%UNI_PHREG (\n\nDATASET = anal,\n\nEVENT = os_surg,\n\nCENSOR = os_censor,\n\nCLIST = surg_app &cat_var_ref,\n\nNLIST = &num_var,\n\nLOGRANK = F,\n\nTYPE3 = T,\n\nPHA = F,\n\nOUTPATH = &dir,\n\nFNAME = Table 4 Univariate association with Overall Survival);\n\nTITLE;\n\n****************************************************************************;\n\nMultivariable model for the binary or time-to-event outcome\n\nWe performed multivariable analysis with a logistic regression model for the 30-day mortality outcome using %LOGREG_SEL (Table 5) and a Cox proportional hazards model for overall survival using %PHREG_SEL (Table 6). The adjusted association of the surgical approaches with the two clinical outcomes was estimated after controlling for observed confounding variables. The odds ratio and hazard ratio were reported along with 95% CI and p-values. In both macros, a manual backward elimination procedure was implemented by dropping one variable at a time until all variables left satisfied a pre-specified alpha level (e.g., SLSTAY = 0.1). The selection process in the macros allows the sample size to adjust and always uses the maximum available sample as the number of variables in the model drops, which differs from the automatic selection procedure built into PROC LOGISTIC or PROC PHREG, where a complete and fixed data set is used throughout the selection procedure. In the following example code, DSN is for specifying the data set name, and EVENT in %LOGREG_SEL is to specify the event of interest, for which the probability will be modeled. VAR is for the list of all variables or terms in the initial model, separated by a space. CVAR is for the list of the categorical variables in VAR with the option to specify reference levels as shown in Table 4. INC = k is set to protect the first k variables in VAR from being eliminated, such as a primary exploratory variable and important confounding variables. In this case study, we want to keep surgical approach in the model as it is the study cohort, which was done by putting surg_app on the first position in VAR and setting INC = 1. Setting CLNUM = T outputs the frequency of categorical variables in the final model. The summary information of the selection procedure was presented in the footnote of the final report table. A separate macro for multivariable analysis of competing risk data is %FINEGRAY_SEL. In reality, there are many approaches to build a multivariable model. If a user wants to customize the model building process, they can use these two macros for reporting purposes only by setting VAR = final model variables selected by other approaches and INC = total number of items in the final model.\n\n Number of observations in the original data set = 34048. Number of observations used = 34048. ** Backward selection with an alpha level of removal of 0.1 was used. The following variables were removed from the model: AJCC Clinical T, Urban/Rural 2003, and Year of Diagnosis.\n\nNumber of observations in the original data set = 34048. Number of observations used = 33150. Backward selection with an alpha level of removal of .10 was used. The following variables were removed from the model: Year of Diagnosis.\n\nIn Table 5, the backward elimination drops clinical T stage, urban/rural and year of diagnosis from the 30-day mortality model at an alpha level of removal of 0.1, which means all variables in the final model have a p-value < 0.1. After controlling for the selected variables in the model, open surgery patients had a 41% higher odds to die within 30-days after surgery comparing to minimally invasive surgery patients (OR = 1.41; 95%CI = (1.18 - 1.69); p < 0.001). In Table 6, year of diagnosis was dropped from the final model to predict overall survival. Patients that underwent open surgery had 12% more chance to die before those that underwent minimally invasive surgery (HR = 1.12; 95% CI = (1.06-1.18); p < 0.001).\n\nStratified Multivariable model\n\nWhen fitting the data into a main-effect model, as in Table 5 or Table 6, an imposed assumption is that the effect of treatment on outcomes is the same across all subgroups defined by the controlled variables. This assumption may or may not hold, and exploring and identifying subgroups that may benefit more from treatment can lead us to a deeper insight. Instead of splitting the dataset into smaller, separated data sets, fitting an interaction model on the entire data set is more appropriate18. As shown in Table 7, we fit a multivariable model including an interaction term between surgical approaches and histology, still implementing the backward elimination procedure. In the related %PHREG_SEL code, the first three terms in VAR were protected from elimination by setting INC = 3, to keep the interaction of interest (surg_apphis_cat) in the model. The macro parameters EFFECT and SLICEBY allow users to specify the variables for the treatment effect and subgroups (the two variables have to be categorical variables for the macro to run correctly). Setting SHORTREPORT = T, only reports the hazard ratio (HR) and the p-value of surgical approach by histology subgroup, and if set to F, the HR for all other control variables in the model will be reported. This macro can only handle one interaction at a time but is useful as an initial exploration for the treatment effect in subgroups.\n\nNumber of observations in the original data set = 34048. Number of observations used = 33150. Backward selection with an alpha level of removal of .10 was used. The following variables were removed from the model: Year of Diagnosis. The estimated stratified treatment effect was controlled by: AJCC Clinical T, Age at Diagnosis, Charlson- Deyo Score, Sex, Urban/Rural 2003.\n\nIn Table 7, detailed information about variable selection in the model building process is shown in the footnote. We see that, overall, minimally invasive surgery shows a protective effect on survival compared to open surgery, but such protection is more pronounced among adenocarcinoma patients (HR = 0.85; p < 0.001) than squamous cell carcinomas (HR = 0.96; p = 0.321). The p-value for interaction term is 0.069, and it tests the difference among the HR of 0.85, 0.95, and 0.96 for the three histology groups. If using the significance level of 0.05, the interaction p-value may confirm that the hazard ratio for the surgical approaches is the same across the histology groups. In the case of a significant interaction effect, researchers should report the interaction model and discuss the treatment effect in each subgroup.\n\n**** SAS® code example for %PHREG_SEL and %LOGREG_SEL ***********;\n\nTITLE 'Table 5 Multivariable Logistic Regression Model for 30-day Mortality';\n\n%LOGREG_SEL(\n\nDSN = anal,\n\nOUTCOME = PUF_30_DAY_MORT_CD,\n\nEVENT = 'Yes',\n\nVAR = surg_app &cat_var &num_var,\n\nCVAR = surg_app&cat_var_ref,\n\nINC = 1,\n\nSLSTAY = 0.1,\n\nCLNUM = F,\n\nOUTPATH = &dir,\n\nFILENAME = Table 5 Multivariable Logistic Regression Model for 30-day Mortality);\n\nTITLE 'Table 6 Multivariable Cox Proportional Hazard Model for Overall Survival';\n\n%PHREG_SEL (\n\nDSN=anal,\n\nEVENT = os_surg,\n\nCENSOR = os_censor,\n\nVAR = surg_app &cat_var &num_var,\n\nCVAR = surg_app * &cat_var_ref ,\n\nINC = 1,\n\nSLSTAY = .10,\n\nCLUNM=T,\n\nOUTPATH = &dir,\n\nFILENAME = Table 6 Multivariable Cox Proportional Hazard Model for Overall Survival);\n\nTITLE 'Table 7 Multivariable Cox Model for OS Stratify by Histology';\n\n%PHREG_SEL(\n\nDSN=anal,\n\nEVENT = os_surg,\n\nCENSOR = os_censor,\n\nVAR = surg_app his_cat surg_apphis_cat &cat_var &num_var,\n\nCVAR = surg_app &cat_var ,\n\nINC = 3,\n\nSLSTAY = .10,\n\nEFFECT = surg_app,\n\nSLICEBY = his_cat,\n\nSHORTREPORT = T,\n\nOUTPATH = &dir,\n\nFILENAME = Table 7 Multivariable Cox Model for OS Stratify by Histology);\n\nTITLE;\n\n**************************************************************************;\n\nKaplan–Meier analysis\n\nIt is standard practice in many time-to-event studies to report Kaplan–Meier (KM) plots, median survival times, and accrual survival rates for time-to-event outcomes stratified by treatments, as a straightforward and intuitive way to assess the survival profile. %KM_PLOT was used to generate Figure 2. The KM plot for overall survival (specified in EVENTS, CENSORS) stratified by surgical approaches (specified in GRPLIST) was carried out with the key information of interest reported in a summary table. This macro has many options that allow the user to control the appearance of the plot (TITLE, XTICK, XMAX, NONCENSORED, and ATRISK) and output the estimated accrual survival rate at pre-specified time points (TIMELIST). This macro becomes handier when there are multiple datasets, several outcomes, or multiple variables of interest. Users can produce multiple KM plots in one macro call for all combinations of the parameter values by listing multiple values separated by spaces in DSN, EVENTS/CENSORS, and GRPLIST.\n\nIn Figure 2, the two survival curves by surgical approaches do not reach the survival probability of 0.5, and hence the median survival time is not estimable (will show as NAs). The 60-month (5-year) survival rate is 68.5% in the minimally invasive surgery group, while 64.2% in the open surgery group. Overall, the log-rank p < 0.001 indicates a significantly improved overall survival in the minimally invasive surgery group over the open surgery group (note that the KM method is only univariate analysis). However, a gain of 4.3% improvement in 5-year survival rate may or may not be clinically significant, and a large sample size may easily lead to small p-values. In such cases, researchers may shed light on both the clinical relevance and statistical significance to interpret results.\n\n******** SAS® code example for %KM_PLOT ******* ***************;\n\n%KM_PLOT(DSN = anal,\n\nEVENTS = os_surg,\n\nCENSORS = os_censor,\n\nGRPLIST = surg_app,\n\nTITLE = \"Figure 2 KM plot of Overall Survival by Surgical Approach\",\n\nUNIT = Month, JOIN = T, PLOT = T, TABLE = T,\n\nTIMELIST = 12 60, NONCENSORED = T,\n\nXTICK = (0 12 24 36 48 60), XMAX = 60, ATRISK = T,\n\nOUTPATH = &dir,\n\nFNAME = Figure 2 KM plot of Overall Survival by Surgical Approaches);\n\n***************************************************************************;\n\n\nDiscussion\n\nA natural course of research is generally iterative and conducted by a research team with mixed expertise and experience levels. The proposed sequential SAS® macros seamlessly fit into that type of research environment. They help process a massive amount of variables effortlessly, produce complete and interpretable summary information to support the research team to comprehend and better plan for the next step, and provide a highly-organized and concise coding interface that facilitates easy updates as research evolves.\n\nResearch based on observational or retrospective data needs extra effort in the study design and data management before data analysis, and careful interpretation afterward. The presented case study also illustrated a simple analytic workflow showing how to build an analytic project from the foundation, comprehend different layers of information jointly from the routine data analyses, and envision where you are and where to go. This work can serve as a nice tutorial for researchers to easily get their research off the ground.\n\nThe limitations include the requirements for researchers to be comfortable with the SAS® environment and have basic statistical training in data handling and interpretation. The proposed SAS® macros don’t handle study design or data management. A properly defined study population, cohort, outcomes, covariates, and sufficient literature review are critical before using our macros for the sake of research quality. Since only relevant information is summarized and reported, the macros don’t cover issues such as assumption check or goodness of fit about the fitted model at this time point. Including an experienced biostatistician in the research team would be beneficial.\n\nThese macros were first created in 201119, and have been implemented in many projects to help turn out high-quality research, efficiently. They are still under continuous upgrade to meet new needs.\n\n\nData availability\n\nOwing to data protection concerns, data used in the use case cannot be shared under the American College of Surgeons' Commission on Cancer NCDB Participant Use File (PUF) Purpose and Terms of Agreement. For information on how to apply for access to NCDB PUF and who will be granted access to it, please visit: https://www.facs.org/quality-programs/cancer/ncdb/puf.\n\n\nSoftware availability\n\nSource code available from: https://github.com/Emory-Yuan/BBISR-SAS-Macros/tree/V1.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.221637713.\n\nLicense: MIT license.", "appendix": "Grant information\n\nResearch reported in this publication was supported in part by the Biostatistics and Bioinformatics Shared Resource of Winship Cancer Institute of Emory University and NIH/NCI under award number P30CA138292.\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 would like to express our sincere gratitude to Yaqi Jia, Sungjin Kim, and Renjiang Jiang for their valuable contributions to those SAS® macros, and their names are shown inside each macro they worked. People who provided strong support and valuable input include Renee H. Moore and Theresa W. Gillespie.\n\n\nReferences\n\nHankey BF, Ries LA, Edwards BK: The surveillance, epidemiology, and end results program: a national resource. Cancer Epidemiol Biomarkers Prev. 1999; 8(12): 1117–1121. PubMed Abstract\n\nBilimoria KY, Stewart AK, Winchester DP, et al.: The National Cancer Data Base: a powerful initiative to improve cancer care in the United States. Ann Surg Oncol. 2008; 15(3): 683–690. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBall R, Robb M, Anderson SA, et al.: The FDA's sentinel initiative--A comprehensive approach to medical product surveillance. Clin Pharmacol Ther. 2016; 99(3): 265–268. PubMed Abstract \| Publisher Full Text\n\nHansen LG, Chang S: Health Research Data for the Real World: The MarketScan Databases. White Paper: TRUVEN Health Analytics. 2011. Reference Source\n\nDreyer NA, Tunis SR, Berger M, et al.: Why observational studies should be among the tools used in comparative effectiveness research. Health Aff (Millwood). 2010; 29(10): 1818–1825. PubMed Abstract \| Publisher Full Text\n\nDreyer NA, Schneeweiss S, McNeil BJ, et al.: GRACE principles: recognizing high-quality observational studies of comparative effectiveness. Am J Manag Care. 2010; 16(6): 467–471. PubMed Abstract\n\nvon Elm E, Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Bull World Health Organ. 2007; 85(11): 867–872. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson ML, Crown W, Martin BC, et al.: Good research practices for comparative effectiveness research: analytic methods to improve causal inference from nonrandomized studies of treatment effects using secondary data sources: the ISPOR Good Research Practices for Retrospective Database Analysis Task Force Report--Part III. Value Health. 2009; 12(8): 1062–1073. PubMed Abstract \| Publisher Full Text\n\nCox E, Martin BC, Van Staa T, et al.: Good research practices for comparative effectiveness research: approaches to mitigate bias and confounding in the design of nonrandomized studies of treatment effects using secondary data sources: the International Society for Pharmacoeconomics and Outcomes Research Good Research Practices for Retrospective Database Analysis Task Force Report--Part II. Value Health. 2009; 12(8): 1053–1061. PubMed Abstract \| Publisher Full Text\n\nBerger ML, Mamdani M, Atkins D, et al.: Good research practices for comparative effectiveness research: defining, reporting and interpreting nonrandomized studies of treatment effects using secondary data sources: the ISPOR Good Research Practices for Retrospective Database Analysis Task Force Report--Part I. Value Health. 2009; 12(8): 1044–1052. PubMed Abstract \| Publisher Full Text\n\nMotheral B, Brooks J, Clark MA, et al.: A checklist for retrospective database studies--report of the ISPOR Task Force on Retrospective Databases. Value Health. 2003; 6(2): 90–97. PubMed Abstract \| Publisher Full Text\n\nSauerbrei W, Abrahamowicz M, Altman DG, et al.: STRengthening analytical thinking for observational studies: the STRATOS initiative. Stat Med. 2014; 33(30): 5413–5432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Y: Emory-Yuan/BBISR-SAS-Macros V1 (Version V1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.2216378\n\nAustin PC: An Introduction to Propensity Score Methods for Reducing the Effects of Confounding in Observational Studies. Multivariate Behav Res. 2011; 46(3): 399–424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrank E, Harrell J: Regression Modeling Strategies. Springer-Verlag; 2006. Reference Source\n\nMedbery RL, Gillespie TW, Liu Y, et al.: Nodal Upstaging Is More Common with Thoracotomy than with VATS During Lobectomy for Early-Stage Lung Cancer: An Analysis from the National Cancer Data Base. J Thorac Oncol. 2016; 11(2): 222–233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFine JP, Gray RJ: A Proportional Hazards Model for the Subdistribution of a Competing Risk. J Am Stat Assoc. 1999; 94(446): 496–509. Publisher Full Text\n\nWang R, Lagakos SW, Ware JH, et al.: Statistics in medicine--reporting of subgroup analyses in clinical trials. N Engl J Med. 2007; 357(21): 2189–2194. PubMed Abstract \| Publisher Full Text\n\nNickleach D, Liu Y, Shrewsberry A, et al.: SAS® Macros to Conduct Common Biostatistical Analyses and Generate Reports. SESUG 2013: The Proceeding of the SouthEast SAS User Group. Reference Source" }	[ { "id": "46844", "date": "26 Apr 2019", "name": "Margaret Stedman", "expertise": [ "Reviewer Expertise Biostatistics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a series of SAS macros that automate a standard analysis of observational data. The macros include a descriptive analysis for table 1. An univariate analysis with the study outcome and study cohort and a multivariable or adjusted analysis. Outcome types include logistic regression, survival analysis, and competing risks.\n\nThe authors do a good job describing the programs and giving examples of the output. My main comment is that there are other macros available to create tables from SAS output and for someone who is adept at SAS the time savings may not be so great. However, it is nice to have them grouped together in a series. I wonder if the authors couldn't go a step further and make the user interface a little more user friendly (such as an ap or point and click) system? I think such system would be more useful to the general public that is not so adept at SAS.\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": "4607", "date": "05 Jun 2019", "name": "Yuan Liu", "role": "Author Response", "response": "We sincerely appreciate Dr. Stedman’s comments. Her suggestion about one more step further capture the trend of the current software development to enhance end-user experience, such as a web base application. I cannot agree more about her vision. Ideally, after most critical steps about study design and data management, data summary should just be one-click away. To relieve from manual data handling would allow to best use human brain power to focus on the data comprehension and critical thinking." } ] }, { "id": "46845", "date": "26 Apr 2019", "name": "Hong-Qiu Gu", "expertise": [ "Reviewer Expertise epidemiology", "biostatistics", "and clinical research in cardiology", "neurology", "and 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\nDr. Yuan Liu and colleagues had developed a series of generic SAS macros aiming to carry out streamlined routine data analyses and produce journal-quality summary tables. Although this work is fundamental, it’ll benefit statisticians a lot by avoiding time-consuming and tedious copy-and-paste processes. The idea of “streamlined routine data analyses” has been in my mind for years, and I do deem that all the available SAS macros in the SAS community are segmented for the whole data analyses process, thus, what we need is not more segmented SAS macros, but a set of SAS macros that can work seamlessly as an entire system. That’s why I developed the ‘%ggTable’ series of SAS macros and published ‘%ggBaseline’, the first macro of the ggTable series (Gu et al., 20181). Although other SAS macros of this series are still under preparation for a peer-reviewed publication, I’m glad to be a reviewer of this work and share some of my considerations that might help move this publication forward.\n\nMajor considerations:\nThe title of this paper might be a little broad or exaggerated. Besides binary and time to event outcomes discussed in this article, multinomial and continuous variables can also be outcomes in observational studies. In addition, many statistical methods, such as propensity score-based methods, say stratifying, matching, and inverse probability weighting, are more and more popular nowadays in observational studies. Last, it’s common to use generalized estimating equation (GEE) models or mixed models to account for clustered data, since many centers are enrolled in observational studies.\n\nMinor considerations:\nFor %DESCRIPTIVE, %UNI_CAT and % UNI_NUM, the variables list is specified by CLIST and NLIST separately; this way has some drawbacks, such as 1) The order in the table will be grouped by variable type and always be categorical variables first; 2) Statistical tests and variable labels cannot be specified when calling the macros. However, this would not be a problem in %ggBaseline.\n\nIt would be better to report the mean with standard deviation (SD) in the same line as mean ± SD, the median with interquartile range (IQR) as median (IQR).\n\nK-M plot with No. at risk is highly recommended, and the border of the legend should be removed as well.\n\nIt would be great if the authors can improve these SAS macros with the consideration of the above concerns. If there is no improvement, more discussions on these limitations and comparison to other available macros are needed in the discussion section.\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": "4606", "date": "05 Jun 2019", "name": "Yuan Liu", "role": "Author Response", "response": "We sincerely appreciate Dr. Gu’s vision and idea which agrees with our very initial motivation to create those macros (Dana et al, 2013, https://analytics.ncsu.edu/sesug/2013/PO-05.pdf), and her creation about %ggTable is elegant and useful to help us improve in the future. For the major consideration, we do have several macros (%UNI_GENMOD, %GENMOD_SEL) that handle the reporting based on the generalized linear regression model (normal, binary, Poisson, negative binomial distribution) for either GEE (clustered data)or non-GEE (independent data) using PROC GENMOD procedure in SAS. In the revision, we added them into the Box 1 with a few more explanation in the context. Even the case study did not use them, they are all available in our final downloadable package, and the usage is similar as in other macros in the case study. For the propensity score-based methods, we also created a series of macros for propensity score estimation, matching/weighting/stratification, balance check, visualization, etc. but since this paper was mainly focus on the routine data analyses, we decided to leave the PS related components and to develop a separate paper in the future. We do appreciate that Dr. Gu pointed out some limitations and have included those limitations in the discussion section. However, we do actively upgrade those macros as we go to meet new needs. Since first created in 2011, some macros have reached their 20th version. We would high appreciate any feedback and comments from the potential users and will improve with your help." } ] } ]	1	https://f1000research.com/articles/7-1955
https://f1000research.com/articles/8-781/v1	04 Jun 19	{ "type": "Case Report", "title": "Case Report: Pigmented paravenous retinochoroidal atrophy: a case report", "authors": [ "Mohammad Zarei", "Raziyeh Mahmoudzadeh", "Hamid Riazi-Esfahani", "Mohammad Zarei", "Raziyeh Mahmoudzadeh" ], "abstract": "This article, to the best of our knowledge, reports the youngest typical case of pigmented paravenous retinochoroidal atrophy (PPRCA) reported to date. A 27-month-old girl presented with exodeviation in her right eye. She had normal birth and development with unremarkable family history. There were no inflammatory signs. In funduscopy, typical bilateral radial paravenous pigmentary changes and retinochoroidal atrophy were noticed in both eyes. The pigmentations consisted of coarse black pigmentations and fine subretinal yellowish round flecks. They arborized into the peripheral retina along the veins. Unaffected areas between the lesions seemed to be normal. Electroretinogram (ERG) responses showed mild to moderate reductions in both scotopic and photopic tests. Based on retinal examination and ERG findings PPRCA was diagnosed. On 16-month follow up, clinical and ERG findings were the same as the initial presentation. This case showed no progression during 16 months of follow up, which may indicate that primary congenital PPRCA with no inflammatory association may be a non-progressive disease.", "keywords": [ "pigmented paravenous retinochoroidal atrophy", "Retinochoroidal atrophy" ], "content": "Introduction\n\nPigmented paravenous retinochoroidal atrophy (PPRCA) is a bilateral and symmetrical condition1,2, which is characterized by atrophy of choriocapillaris and retinal pigment epithelium (RPE), and pigmentation along the retinal veins3. Patients are usually asymptomatic and diagnosis is made during routine examination based on typical fundus appearance and non-progressive nature of disease2. Here we present, to the best of our knowledge, the youngest typical case of PPRCA reported to date.\n\n\nCase report\n\nA 27-month-old girl presented with exodeviation in his right eye to retina clinic, at Farabi eye hospital in April 2016. She experienced a normal birth and development, and had unremarkable family history. She used no medications. Visual acuity testing was not feasible due to patient’s young age. Her parents had noticed outward deviation in her right eye. The cyclorefraction of both eyes were +2.25 diopters. The anterior segment and vitreous examination were normal and there were no inflammatory signs. In funduscopy, typical bilateral radial paravenous pigmentary changes and retinochoroidal atrophy were noticed in both eyes (Figure 1). The pigmentations consisted of coarse black pigmentations and fine subretinal yellowish round flecks. They arborized into the peripheral retina along the veins. Unaffected areas between the lesions seemed to be normal.\n\nThese pigmentary changes were arborized into the peripheral retina along the veins. Unaffected areas between the lesions appeared normal.\n\nThe fundus examination of her parent and newly born brother showed no abnormality. Electroretinogram (ERG) responses showed mild to moderate reduction in both scotopic and photopic responses, equally. Based on retinal examination and ERG findings, PPRCA was diagnosed. At 9 months later, with no treatment, the fundus findings showed no changes and cyclorefraction was +1 diopter. On 16-month follow up, fundoscopy and ERG findings were the same as those at the initial presentation.\n\n\nDiscussion\n\nTo the best of authors’ knowledge, this patient is the youngest with PPRCA ever reported. The patient showed typical characteristics of PPRCA and considerable stability over the course of a 16-month follow up. This presentation is in concordance with the congenital origin hypothesis of PPRCA3,4. In 1937, Brown described this condition for the first time in a 47-year-old man with alopecia areata and named it as retinochoroiditis radiata. The patient had already been under treatment for a disseminated choroiditis at the age of 26 years and had symptoms of tuberculous spondylitis. Considering that his close relatives had died of tuberculosis, the condition was presumed to be a form of tuberculous periphlebitis5,6. The hypothesis of inflammatory origin was further confirmed by other case reports with congenital syphilis, rubeola, measles and Behçet's disease. However, in the years after, a hypothesis of congenital origin was developed and the condition was considered a hereditary disease4,7.\n\nLater in 1962 Franceschetti, changed the name of this condition from retinochoroiditis radiata to a more generally accepted term of PPRCA4,8. So far, there are more than 100 case reports in literature and most of the affected patients are men3,4. In 2003, after lengthy follow ups, Yanagi et al.9, found that this disease is stationary in younger patients while is slowly progressive in older subjects. The progression in older patients may be attributed to wrong diagnosis of PPRCA and these patients may actually have pseudo-PPRCA.\n\nAlthough the majority of cases have been sporadic, there are reports of familial occurrence2,3. In 1986, Traboulsi and Maumenee10 described a mother and her three sons with PPRCA. Every member had a different chief complaint. The youngest son. who was 4 years old. had poor fixation, nystagmus, and peripheral pigmentary abnormalities. The 10-year-old son had no signs of pigmentary change and the second son had mild pigmentary changes at age of 7 years. To our knowledge, the members of this family were the youngest cases ever been reported to date.\n\nERG findings are not the same in all cases and have a wide spectrum. While in some cases, ERG shows noticeable involvement, in others it may be normal or show only mild involvement. Reduction of b wave amplitude is the most common finding, followed by a wave amplitude reduction and prolonged latency3,4,7. In some cases, rod responses may be affected more than cone responses, while in others cone response reduction is the dominant feature. In our case both responses were almost equally diminished. This variation may reflect the heterogeneous impairment of various cell types in the retina4.\n\nOur patient presented with exotropia in right eye and bilateral fundus involvement. Previous studies have reported the association of PPRCA with different ocular problems, including anisometropia, amblyopia, esotropia, exotropia, nystagmus, optic disc drusen, and macular changes, such as cystoid macular edema, pigmentary macular degeneration, lamellar macular holes and macular coloboma3,4. Since secondary PPRCA or pseudo-PPRCA has been reported, clinicians should be aware of differential diagnoses, which include chorioretinal degenerations, serpiginous choroidopathy, retinits pigmentosa, tuberculous disseminated choroiditis, helicoid peripapillary chorioretinal atrophy and angioid streaks7,9,11,12.\n\nOne of the main advantages of this case report was a relatively long-term follow-up period. This case showed no progression during 16 months of follow up, which may indicate that primary congenital PPRCA with no inflammatory association may be a non-progressive disease. Cases with progressive course or other concomitant findings may be secondary PPRCA and pseudo-PPRCA can be a better term for them.\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 parents of the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNoble KG, Carr RE: Pigmented paravenous chorioretinal atrophy. Am J Ophthalmol. 1983; 96(3): 338–344. PubMed Abstract \| Publisher Full Text\n\nShona OA, Islam F, Robson AG, et al.: PIGMENTED PARAVENOUS CHORIORETINAL ATROPHY: Detailed Clinical Study of a Large Cohort. Retina. 2019; 39(3): 514–529. PubMed Abstract \| Publisher Full Text\n\nTsang SH, Sharma T: Pigmented Paravenous Chorioretinal Atrophy (PPCRA). Adv Exp Med Biol. 2018; 1085: 111–113. PubMed Abstract \| Publisher Full Text\n\nHuang HB, Zhang YX: Pigmented paravenous retinochoroidal atrophy (Review). Exp Ther Med. 2014; 7(6): 1439–1445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTraversi C, Tosi GM, Caporossi A: Unilateral retinitis pigmentosa in a woman and pigmented paravenous chorioretinal atrophy in her daughter and son. Eye (Lond). 2000; 14(Pt 3A): 395–397. PubMed Abstract \| Publisher Full Text\n\nMurray AT, Kirkby GR: Pigmented paravenous retinochoroidal atrophy: a literature review supported by a unique case and insight. Eye (Lond). 2000; 14(Pt 5): 711–716. PubMed Abstract \| Publisher Full Text\n\nTandon M, Shukla D, Huda R, et al.: Pigmented paravenous chorioretinal atrophy with Coat's like response. Indian J Ophthalmol. 2013; 61(10): 586–588. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranceschetti A: A curious affection of the fundus oculi: helicoid peripapillar chorioretinal degeneration. Its relation to pigmentary paravenous chorioretinal degeneration. Doc Ophthalmol. 1962; 16: 81–110. PubMed Abstract \| Publisher Full Text\n\nYanagi Y, Okajima O, Mori M: Indocyanine green angiography in pigmented paravenous retinochoroidal atrophy. Acta Ophthalmol Scand. 2003; 81(1): 60–67. PubMed Abstract \| Publisher Full Text\n\nTraboulsi EI, Maumenee IH: Hereditary pigmented paravenous chorioretinal atrophy. Arch Ophthalmol. 1986; 104(11): 1636–1640. PubMed Abstract \| Publisher Full Text\n\nHashimoto Y, Kase S, Saito W, et al.: Abnormalities of fundus autofluorescence in pigmented paravenous chorioretinal atrophy. Open Ophthalmol J. 2012; 6: 125–128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernández-Da Mota SE, Chacón-Lara A: Bilateral pigmented paravenous chorioretinal atrophy: a case report. Case Rep Ophthalmol. 2011; 2(2): 228–231. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "49470", "date": "24 Jun 2019", "name": "Kim Ramasamy", "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\nWhat is the name of the imaging device/fundus camera?\n\nCan we get the periphery photo? Any history of laser photocoagulation done? The current figure shows 2 pigmented spots at the left margin.\n\nOther eye Fundus photo?\n\nERG report available? Please provide the report or at least the important line graphs e.g scotopic, combined response and photopic response.\n\nIn the case report it is described that: \"The pigmentations consisted of coarse black pigmentations and fine subretinal yellowish round flecks. They arborized into the peripheral retina along the veins\". However practically, the abnormal pigmentation is a hypopigmentation of the affected area of fundus.\n\nIn the discussion section, there is no need to start with “the best of authors’ knowledge, this patient is the youngest with PPRCA ever reported.” This can be written at the end.\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": [] }, { "id": "62575", "date": "23 Apr 2020", "name": "Maria Prieto del Cura", "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 review in my opinion will be approved with reservations to improve the paper I will make some suggestions:\nPicture of fundus of both eyes. Is it possible Autofluorescence or AFG?\n\nWhat kind of Electroretinogram has been made? Picture of it to show the response.\n\nDifferential Diagnosis, 27-month-old girl is too young to think in PPRA, what kind of systemic or genetic diagnosis has been made to get to that excluded diagnosis?\n\nAsk for pregnant infectious illness: Rubeola, Toxoplasma....\nI have doubts that the diagnosis will be correct with the information provided in the paper.\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? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-781
https://f1000research.com/articles/8-780/v1	04 Jun 19	{ "type": "Data Note", "title": "Attention Network Test fMRI data for participants with Parkinson’s disease and healthy elderly", "authors": [ "Trevor K. M. Day", "Tara M. Madhyastha", "Mary K. Askren", "Peter Boord", "Thomas J. Montine", "Thomas J. Grabowski", "Tara M. Madhyastha", "Mary K. Askren", "Peter Boord", "Thomas J. Montine", "Thomas J. Grabowski" ], "abstract": "Here, we present unprocessed and preprocessed Attention Network Test data from 25 adults with Parkinson’s disease and 21 healthy adults, along with the associated defaced structural scans. The preprocessed data has been processed with a provided Analysis of Functional NeuroImages afni_proc.py script and includes structural scans that were skull-stripped before defacing. All acquired demographic and neuropsychological data are included.", "keywords": [ "Attention Network Task", "ANT", "fMRI", "Parkinson’s disease", "attention" ], "content": "Introduction\n\nAttention dysfunction is a common symptom of Parkinson’s disease (PD) and has a significant impact on quality of life. Approximately half of all people with PD suffer from attention and/or memory symptoms (Barone et al., 2009).\n\nThe data included here is a subset of data from a study (Cholerton et al., 2013) that used the Attention Network Test (ANT) (Fan et al., 2005) to measure three aspects of attention: alerting (achieving and maintaining an alert state), orienting (selecting the spatial location of sensory input), and executive control (resolving conflict). We acquired structural and functional MRI images at two occasions in participants with and without PD, with six randomly ordered repetitions of the ANT task (labeled 1–6) at each occasion. Each numbered run represents the same stimulus list between subjects, although the six runs were presented to each subject in a different order.\n\nData described in this paper have previously been analyzed in Boord et al. (2017) and Madhyastha et al. (2015), wherein the runs were labeled A-F rather than 1–6.\n\n\nMaterials and methods\n\nProcedures were approved by the University of Washington Institutional Review Board (#41304) and subjects provided written informed consent.\n\nThe sample of subjects includes 25 participants with PD and 21 healthy controls (HC) who participated in two scanning sessions, which were one to three weeks apart. PD participants were recruited from a larger parent study where they underwent extensive clinical examination and neuropsychological assessment (Cholerton et al., 2013).\n\nDemographic information is provided in Table 1. PD and HC participants did not differ on age (t(40) = 1; p = 0.2) or years of education (t(40) = 0.6, p = 0.6), but did differ on the Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS; Goetz et al., 2007) part III (t(30) = 10; p < .001). Participants also underwent a battery of neuropsychological tests (Cholerton et al., 2013). Neuropsychological test results are provided in Table 2. PD and HC participants did not differ on any of the cognitive tests that were administered to both groups. HC participants obtained only a subset of the measurements.\n\nParticipants with PD and healthy controls did not differ on age, UPDRS III, or years of education.\n\nControls are included where they were administered the exam.\n\nAbbreviations: Boston Visual Retention Test (BVRT); Judgment of Line Orientation (JLO); Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA); Weschler Adult Intelligence Scale (WAIS)\n\nOne subject (RC4206) had an acquisition error during their second session structural scan. Correspondingly, their structural scan from their first session has been copied for their second session to create a valid Brain Imaging Data Structure (BIDS) directory.\n\nAt each of the two sessions, we acquired six repetitions of the task and T1-weighted structural images from each subject. Data were acquired using a Philips 3.0T X-Series Achieva MR System (Philips Medical Systems, software version R2.6.3) with a 32-channel SENSE head coil. Each session included functional and structural scans. For task scans, whole-brain axial echo-planar images (43 sequential ascending slices, 3 mm isotropic voxels, field of view = 240 x 240 x 129 mm, repetition time = 2400 ms, echo time = 25 ms, flip angle = 79°, SENSE acceleration factor = 2) were collected parallel to the AC-PC line. Each functional scan was 149 volumes (5.96 min). A sagittal T1-weighted 3D MPRAGE (176 slices, matrix size = 256 x 256, inversion time = 1100 ms, turbo-field echo factor = 225, repetition time = 7.46 ms, echo time = 3.49 ms, flip angle = 7°, shot interval = 2530 ms) with 1 mm isotropic voxels was also acquired for registration and tissue analyses.\n\nIn total, 45 subjects completed all six task scans in both sessions. One subject did not complete the second session; and one subject is missing task data for the first four task scans (out of six) at the second session.\n\nMost scans were available in the Digital Imaging and Communications in Medicine (DICOM) file format; and were converted to the Neuroimaging Informatics Technology Initiative (NIfTI) file format using the Analysis of Functional NeuroImages (AFNI) program dcm2niix_afni. Subjects with missing DICOMs had Philips format PAR/RECs available and were also converted to NIfTI format using AFNI dcm2niix_afni (Day et al., 2019).\n\nWe used the ANT (Fan et al., 2005; Fan et al., 2002), which combines cues and targets within a single reaction time task to measure the efficiency of the alerting, orienting, and executive attention networks. Each session included six separate task runs. Each run included two buffer trials followed by 36 reaction time trials (a total of 432 trials per subject).\n\nA full description of the ANT can be found in Fan et al. (2005). Briefly, in the ANT, subjects are asked to determine the direction of an arrow (left or right); which is flanked by four other arrows. These flanker arrows either point the same direction as the probe arrow (“congruent”) or the opposite direction (“incongruent”). The row of arrows appears either above or below the center of the screen, and prior to displaying the arrows, the participants are presented with a) no cue; b) a spatial cue that reflects where the arrows will appear; or c) a center cue. A fixation cross appeared throughout the trial.\n\nfMRI data were preprocessed using AFNI (Cox, 1996), version AFNI_17.3.00 (Oct. 12, 2017). Processing steps were generated with afni_proc.py (version 5.18, Sept. 12, 2017), treating each repetition of the ANT task as a single scan (i.e. no concatenation).\n\nafni_proc.py call\n\nFirst four parameters are set on a per-subject basis and represented here with asterisks (*).\n\n\n\nWe used the following blocks: despike, tshift (default), align, tlrc, volreg (default), blur (default), regress (default). Frames were despiked and slice-timing corrected (tshift). During the align stage, we aligned the functional to the structural using the lpc+ZZ cost function. Following structural alignment, we aligned the data to the Montreal Neurological Institute (MNI) 152 standard space (2009c) template, and the data was blurred with a 4 mm full-width half-max filter and masked using 3dAutomask algorithms. Frames were registered to the minimum outlier and then aligned to standard space. We used anaticor (Jo et al., 2010) to regress out the white matter signal and remove the effects of motion. The final result of the AFNI processing was converted to NIFTI using AFNI 3dAFNIto NIFTI. All scans completed AFNI processing.\n\nThe anatomical scans were defaced using pydeface before organizing in BIDS format. Skull-stripping and registration were performed on the undefaced anatomical scans.\n\nAll code is available on GitHub (Day, 2019).\n\nData are organized according to the Brain Imaging Data Structure (BIDS) (Gorgolewski et al., 2016). All 47 subjects have two sessions, with corresponding func/ and anat/ directories.\n\nThe AFNI-processed data are included in derivatives, matching the format of Nifti/. Also included for convenience are skull-stripped anatomical images, as skull-stripping is known to occasionally fail on defaced images.\n\nFinally, individual scans have matching JSON files in both datasets, created by dcm2niix_afni. Supplementing these files are higher level JSON files (following the naming convention task-ANT?_bold.json) that supply the “TaskName” and “SliceTiming” parameters. Slice timing information is required by the BIDS format, and as the pre-processed (“derivatives”) data has been slice-timing corrected, an array of zeros is provided for this field.\n\nTask timing data are included on the scan level. The “onset” and “duration” columns are in seconds, and the “trial_type” column includes cue events (“CenterCue,” “SpatialCue,” “NoCue”), target events (“Congruent,” “Incongruent”), and cue/target errors (“CueErr,” “TargetErr”). Only correct-response trials are included. Errors are also generated when the subject responded too early or not at all.\n\nThe processing script (afniscript.sh) and demographic information (demographics.csv) are included at the top level.\n\n\nData availability\n\nOpenNeuro: ANT: Healthy aging and Parkinson’s disease. https://doi.org/10.18112/openneuro.ds001907.v2.0.3 (Day et al., 2019)\n\nThis project contains the following underlying data:\n\n- sub-RC4101/ – sub-RC4227/ (scans of the 46 participants at two sessions each)\n\nThese folders each contain the following underlying data:\n\n- ses-1/anat (T1w.json and defaced T1w.nii.gz files for session 1)\n\n- ses-1/func (bold.json, bold.nii.gz and events.tsv files for runs 1–6 of session 1)\n\n- ses-2/anat (T1w.json and defaced T1w.nii.gz files for session 2)\n\n- ses-2/func (bold.json, bold.nii.gz and events.tsv files for runs 1–6 of session 2)\n\nOpenNeuro: ANT: Healthy aging and Parkinson’s disease. https://doi.org/10.18112/openneuro.ds001907.v2.0.3 (Day et al., 2019)\n\nThis project contains the following extended data:\n\n- .bidsignore (file to suppress BIDS naming warning messages)\n\n- afniscript.sh (processing script)\n\n- dataset_description.json (BIDS dataset parameters)\n\n- demographics.csv (demographic information for participants)\n\n- README (README file, including changelog)\n\n- task-ANT_bold.json (acquisition parameters for task scan)\n\n- derivatives/ (AFNI-processed functional images within func/ directories; skull-stripped anatomical images within anat/)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\n- Source code available from: https://github.com/IBIC/UdallANT\n\n- Archived source code at time of publication: https://doi.org/10.5281/zenodo.2847832 (Day, 2019)\n\n- License: MIT", "appendix": "Grant information\n\nThis research was supported by NIH RC4 NS073008 (PI: Grabowski), P50 NS062684 (PI: Montine). Peter Boord received postdoctoral support under the Ruth L. Kirschstein National Research Service Award, T32AG0000258.\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 are grateful to the participants of the Pacific Udall Center for contributing their time and data to advance Parkinson’s research.\n\n\nReferences\n\nBarone P, Antonini A, Colosimo C, et al.: The PRIAMO study: A multicenter assessment of nonmotor symptoms and their impact on quality of life in Parkinson's disease. Mov Disord. 2009; 24(11): 1641–1649. PubMed Abstract \| Publisher Full Text\n\nBoord P, Madhyastha TM, Askren MK, et al.: Executive attention networks show altered relationship with default mode network in PD. Neuroimage Clin. 2017; 13: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCholerton BA, Zabetian CP, Quinn JF, et al.: Pacific Northwest Udall Center of excellence clinical consortium: study design and baseline cohort characteristics. J Parkinsons Dis. 2013; 3(2): 205–214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCox RW: AFNI: software for analysis and visualization of functional magnetic resonance neuroimages. Comput Biomed Res. 1996; 29(3): 162–173. PubMed Abstract \| Publisher Full Text\n\nDay TKM, Madyastha TM, Boord P, et al.: Udall Pilot (ANT). OpenNeuro. 2019. http://www.doi.org/10.18112/openneuro.ds001907.v2.0.3\n\nDay TKM: 'ANT: Healthy aging and Parkinson's disease' processing script (Version 1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2847832\n\nFan J, McCandliss BD, Fossella J, et al.: The activation of attentional networks. NeuroImage. 2005; 26(2): 471–479. PubMed Abstract \| Publisher Full Text\n\nFan J, McCandliss BD, Sommer T, et al.: Testing the efficiency and independence of attentional networks. J Cogn Neurosci. 2002; 14(3): 340–347. PubMed Abstract \| Publisher Full Text\n\nGoetz CG, Fahn S, Martinez-Martin P, et al.: Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS): Process, format, and clinimetric testing plan. Mov Disord. 2007; 22(1): 41–47. PubMed Abstract \| Publisher Full Text\n\nGorgolewski KJ, Auer T, Calhoun VD, et al.: The brain imaging data structure, a format for organizing and describing outputs of neuroimaging experiments. Sci Data. 2016; 3: 160044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJo HJ, Saad ZS, Simmons WK, et al.: Mapping sources of correlation in resting state FMRI, with artifact detection and removal. NeuroImage. 2010; 52(2): 571–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadhyastha TM, Askren MK, Boord P, et al.: Dynamic connectivity at rest predicts attention task performance. Brain Connect. 2015; 5(1): 45–59. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "49476", "date": "19 Jun 2019", "name": "Brian Berman", "expertise": [ "Reviewer Expertise Parkinson's disease", "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\nThe authors have done a nice job presenting their imaging data that are being made available for public download. The article is well written and concise and provides background information necessary to enable the utilization of these data by other investigators. A sampling of imaging data provided online was looked at and appears to be appropriate and of good quality. The accompanying demographics file was reviewed and contains pertinent data. One issue I noticed on quick review is that there are MoCA and MMSE scores that exceed 30. Otherwise I have largely minor suggestions in order to improve the accessibility and utilization of these data by others:\nPlease state reason for repeating scanning sessions. If time between two sessions may be relevant for analysis, then please provide timing log for each subject. Would include sex difference comparison between groups. They look like they may be different. BIDS in Materials section could be referenced as is done later under “Organization”. Would use “MDS-UPDRS” in Table 1 to distinguish values from the “UPDRS” scale. Tables should note if parentheses represent standard deviations. (Parentheses are incorrectly used to note units.) There were 149 volumes resulting in 357.6 sec of scanning time. Were the scans actually 6 min long? Were there any dummy scans at begging for T1 equilibration and if so have these been removed? Would explicitly state matrix size and thickness for EPI scans. Was there any gap? Under “Organization” do not need to define BIDS again. Also the sentence about “skull-stripped anatomical images” being included is confusing as all images provided are the defaced images.\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": "62815", "date": "11 May 2020", "name": "Yong Jeong", "expertise": [ "Reviewer Expertise Neuroimaging in neurological 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 data note is on the brain fMRI data of patients with Parkinson’s disease in Openneuro.\nThey provide unprocessed and also preprocessed fMRI data from 25 patients and 21 healthy controls acquired during the attention network test tasks along with T1 structural MRI. The MRI acquisition parameters and preprocessing codes are also provided thus other researchers can replicate the results or use them for other purposes such as machine learning-based classification. They also provide the basic demographics and the performance of cognitive tests.\nGiven increased activities of neuroimage data sharing, the data has values of providing unique fMRI data from patients. There are available task fMRI data from normal controls, however, limited data from patients with neurological diseases or psychiatric disorders. Furthermore, patients with Parkinson’s disease showing motor problems have difficulties in performing taskㄴ in the scanner and commonly have motion artifacts.\nThere are some items needed for the wide use of the data. First, the authors need to provide the medication history of the patients, at least the levodopa dose equivalency. Since, the medication influence a lot on the motor and cognitive performance in the patients and also on brain activities, one may use them as covariates depending on their interests. Second, they need to provide the performance of ANT task corresponding task scan. One can separate sessions into correct or fail, omit, or commit. This approach is popular in the attention fMRI experiment. Third, it will be great if they can provide resting fMRI and/or DTI data. These data can be used for investigating the functional/structural connectivity or network change in the disease.\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": [] } ]	1	https://f1000research.com/articles/8-780
https://f1000research.com/articles/8-779/v1	04 Jun 19	{ "type": "Case Report", "title": "Case Report: A rapidly growing cyst on the scalp", "authors": [ "Emma Short", "Aisling O'Shea", "Krishna Mukkanna", "Girish Patel", "Stefan Docjinov", "Kenneth May", "Aisling O'Shea", "Krishna Mukkanna", "Girish Patel", "Stefan Docjinov", "Kenneth May" ], "abstract": "Trichilemmal carcinoma is a rare tumour derived from the outer root sheath of hair follicles. It can be difficult to distinguish both clinically and histologically from other skin lesions, particularly squamous cell carcinoma. We present the case of a 62-year-old female with a 20-year history of three 1-cm cysts on her scalp. Over a six-month period, a cyst overlying the occiput had become painful and grown in size. The general practitioner and subsequently local emergency department suspected infection. The lesion was incised, and the patient was treated with oral antibiotics. At the time of surgical excision, the lesion measured 3 x 4 cm.\n\nMicroscopic examination identified rounded dermal lobules of squamous epithelium with trichilemmal keratinization, in keeping with a pre-existing pilar cyst. There were areas with nuclear pleomorphism, mitoses and an infiltrative architecture. A diagnosis of trichilemmal carcinoma arising in a pilar cyst was made. Trichilemmal carcinomas are considered to be a low-grade tumour, but they have the potential to spread to lymph nodes and to metastasise to distant sites in the body, therefore adequate excision and appropriate follow-up are required.", "keywords": [ "Trichilemmal carcinoma", "pilar cyst" ], "content": "Introduction\n\nTrichilemmal carcinoma is a rare tumour derived from the outer root sheath of hair follicles1. It typically occurs in elderly patients on sun-exposed areas of the body1. Such tumours may occur de novo, but more commonly they arise from trichilemmal cysts, which are benign lesions arising from the isthmus of hair follicles, or proliferating trichilemmal tumours2. It is thought that trauma and inflammation can induce the transformation of a benign tumour into a malignant tumour2. The tumour may have a prolonged benign period before cancer develops.\n\nThis case report is important as it illustrates that a diagnosis of trichilemmal carcinoma is often delayed due to it mimicking other skin lesions.\n\n\nCase report\n\nA 62-year-old Caucasian British female presented with a 20-year history of three 1-cm cysts on her scalp. She was previously fit and well and had no significant medical history. Over a seven-month period, the cyst overlying the occiput had become painful and grown in size. During this time, the patient had visited her general practitioner and local emergency department, both of which suspected infection. The lesion was incised, and the patient was treated with three courses of oral flucloxacillin (each course, 500 mg four times per day for 1 week) and one course of oral clarithromycin (250 mg twice per day for 1 week). At the time of surgical excision, the lesion measured 3 x 4 cm. It was raised, indurated, crusted, demonstrated a sparsity of hairs on the surface, had superficial ulceration and exuded serosanguinous fluid when pressed (Figure 1). There was no palpable lymphadenopathy.\n\nMicroscopic examination of the lesion identified rounded dermal lobules of squamous epithelium with trichilemmal keratinisation in keeping with a pre-existing pilar cyst (Figure 2A). Areas with nuclear pleomorphism, mitoses and an infiltrative architecture were noted, and they retained trichilemmal keratinisation (Figure 2B–D). The features were of a trichilemmal carcinoma arising in a pilar cyst.\n\n(A) Squamous epithelium with trichilemmal keratinisation (x4 objective). (B–D) Epithelium with nuclear pleomorphism, mitoses and an infiltrative architecture (x20 objective).\n\nShe was reviewed three months post-operatively. The wound had healed well and there was no sign of recurrence.\n\n\nDiscussion\n\nTrichilemmal carcinomas can be difficult to distinguish clinically and histologically from other skin lesions, particularly squamous cell carcinoma (SCC). Microscopically they are characterised by an abrupt transition of nucleated squamous epithelial cells to keratinised cells, without the formation of a granular layer3, and a lobular proliferation of epithelial cells which exhibit nuclear pleomorphism, prominent mitotic activity and infiltration beyond the basement membrane4.\n\nTrichilemmal carcinomas are considered to be a low-grade tumour, but they do have the potential to spread to lymph nodes and to metastasise to distant sites in the body5. There are also reports of death due to the disease6. Therefore, prompt treatment is necessary to reduce morbidity and mortality. Surgical excision with a 1-cm border is the recommended treatment. However, in recent years, Mohs surgery has been used with success.\n\nFor recurrent disease, or cases with lymph node or distant metastases, radiotherapy and chemotherapy are sometimes considered, but often there is no standard protocol for trichilemmal carcinoma treatment, and regimens similar to those used for SCC are employed. Following treatment, patients will need to undergo regular follow-up due to the risk of recurrence and/or metastases. Because of the tumour’s rarity, standard treatment and follow up protocols have not been established.\n\n\nConclusion\n\nTrichilemmal carcinoma is a rare adnexal tumour. It can mimic common skin lesion such as cysts or squamous cell carcinoma. Diagnosis is dependent on microscopic examination, and the identification of features including the absence of a granular cell layer, a lobular architecture, cellular pleomorphism, mitoses and invasion beyond the basement membrane. The tumour can behave aggressively. Adequate excision and appropriate follow-up are required.\n\n\nLearning points\n\nTrichilemmal carcinoma is a rare adnexal tumour.\n\nIt can mimic common skin lesion such as cysts or squamous cell carcinoma.\n\nDiagnosis is dependent on microscopic examination, and the identification of features including the absence of a granular cell layer, a lobular architecture, cellular pleomorphism, mitoses and invasion beyond the basement membrane.\n\nThe tumour can behave aggressively. Adequate excision and appropriate follow-up are required.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nReis JP, Tellechea O, Cunha MF, et al.: Trichilemmal carcinoma: review of 8 cases. J Cutan Pathol. 1993; 20(1): 44–9. PubMed Abstract \| Publisher Full Text\n\nBrownstein MH, Arluk DJ: Proliferating trichilemmal cyst: a simulant of squamous cell carcinoma. Cancer. 1981; 48(5): 1207–14. PubMed Abstract \| Publisher Full Text\n\nKim UG, Kook DB, Kim TH, et al.: Trichilemmal Carcinoma from Proliferating Trichilemmal Cyst on the Posterior Neck. Arch Craniofac Surg. 2017; 18(1): 50–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSau P, Graham JH, Helwig EB: Proliferating epithelial cysts. Clinicopathological analysis of 96 cases. J Cutan Pathol. 1995; 22(5): 394–406. PubMed Abstract \| Publisher Full Text\n\nLobo L, Amonkar AD, Dontamsetty VV: Malignant Proliferating Trichilemmal Tumour of the Scalp with Intra-Cranial Extension and Lung Metastasis-a Case Report. Indian J Surg. 2016; 78(6): 493–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhuang SM, Zhang GH, Chen WK, et al.: Survival study and clinicopathological evaluation of trichilemmal carcinoma. Mol Clin Oncol. 2013; 1: 499–502. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "49455", "date": "02 Jul 2019", "name": "Laszlo Igali", "expertise": [ "Reviewer Expertise Dermatopathology", "melanocytic disorders and skin cancer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article highlights the difficulties in the clinical and histological diagnosis of a rare adnexal tumour. The number of properly described bona fide trichilemmal carcinomas is low, therefore case reports describing the observed histological features are valuable. The authors also highlighted the long clinical history related to a pre-existing pilar cyst - transformation of the tumour can occur, sometimes in a long and unpredictable clinical course.\nI would suggest one area, which could be expanded more is the differential diagnosis (perhaps use and differentiating value of immunohistochemistry) as well as it's relationship to the follicular/infundibular squamous cell carcinoma.\nI would recommend indexing this article as it is - the short and succinct description and important clinical and histological points make this useful, and the point raised above is probably beyond the scope of a case report.\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": "64756", "date": "29 Jun 2020", "name": "Toshitsugu Nakamura", "expertise": [ "Reviewer Expertise diagnostic pathology", "immunohistochemistry", "pathology of cell death and terminal differentiation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a rare case of trichilemmal carcinoma (TC). It may be valuable, from educational aspect, for the practitioners who have not encountered such a rare tumor, with a condition that pathological diagnosis is accurate and strict. It seems to me, however, that Figure 2 does not show characteristic features for TC or pilar cyst (PC). Although the microphotographs in Figure 2 are somewhat out of focus and are not discernible enough, the tumor looks squamous cell carcinoma (SCC). I suggest the authors to present characteristic histopathological features for TC/PC, as follows:\nFigure 2A:\nLow-power view showing cystic structure\n\nDistinct feature of trichilemmal keratinization.\nFigures 2B/2C/2D:\n\nLow-power view showing folliculo-centric structure of the tumor\n\nDistinct feature of trichilemmal keratinization (Absence of granular cell layer is not a warrant of trichilemmal keratinization, because conventional SCC usually shows keratinization from squamoid tumor cells without interposition of granular cell layer)\n\nThe tumor cells with clear cytoplasm, stained by periodic acid-Schiff (PAS) procedure.\nIn case that the present tumor is difficult to distinguish from SCC, immunohistochemical markers for trichilemmal differentiation, such as CD34 and CK17, should be examined and presented.\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? No", "responses": [] }, { "id": "76140", "date": "15 Dec 2020", "name": "Manas Dave", "expertise": [ "Reviewer Expertise Histopathology" ], "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\nIntroduction Well written introduction acknowledging trichilemmoma and its malignant variation.\n\nCase report Grammatical considerations - General Medical Practitioner should be used instead of General Practitioner Well documented photos and descriptions; are higher resolution histology images available?\n\nDiscussion Well written - the authors may wish to add that this case report exemplifies the importance of a comprehensive microscopic examination for seemingly clinically benign pilar cysts and the importance of a thorough histological assessment. This can also be added into the learning points.\n\nConsent from the patient acknowledged\nThis is a well written case report by the authors presenting a uncommon malignancy.\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/8-779
https://f1000research.com/articles/7-1184/v1	03 Aug 18	{ "type": "Research Article", "title": "Factors influencing malignant mesothelioma survival: a retrospective review of the National Mesothelioma Virtual Bank cohort", "authors": [ "Waqas Amin", "Faina Linkov", "Douglas P. Landsittel", "Jonathan C. Silverstein", "Michael J. Becich", "Faina Linkov", "Douglas P. Landsittel", "Jonathan C. Silverstein", "Michael J. Becich" ], "abstract": "Background: Malignant mesothelioma (MM) is a rare but deadly malignancy with about 3,000 new cases being diagnosed each year in the US. Very few studies have been performed to analyze factors associated with mesothelioma survival, especially for peritoneal presentation. The overarching aim of this study is to examine survival of the cohort of patients with malignant mesothelioma enrolled in the National Mesothelioma Virtual Bank (NMVB).\n\nMethods: 888 cases of pleural and peritoneal mesothelioma cases were selected from the NMVB database, which houses over 1400 cases that were diagnosed from 1990 to 2017. Kaplan Meier’s method was performed for survival analysis. The association between prognostic factors and survival was estimated using Cox Hazard Regression method and using R software for analysis. Results: The median overall survival (OS) rate of all MM patients, including pleural and peritoneal mesothelioma cases is 15 months (14 months for pleural and 31 months for peritoneal). Significant prognostic factors associated with improved survival of malignant mesothelioma cases in this NMVB cohort were below the age of 45, female gender, epithelioid histological subtype, stage I, peritoneal occurrence, and had treatment that consisted of combining surgical therapy with chemotherapy. Combined surgical and chemotherapy treatment was associated with improved survival of 23 months in comparison to single line therapies. Conclusions: There has not been improvement in the overall survival for patients with malignant mesothelioma over many years with current available treatment options. Our findings show that combined surgical and chemotherapy treatment is associated with improved survival compared to local therapy alone.", "keywords": [ "Mesothelioma", "Survival analysis. Cox hazard regression analysis", "Biobanking", "Risk factor" ], "content": "Introduction\n\nMalignant mesothelioma is a rare and fatal malignancy, associated with occupational and environmental exposure to asbestos. As per American Cancer Society, approximately 3000 new cases are diagnosed per year in the United States. The pleura is the primary site of mesothelioma occurrence, but it also occurs at other sites (pericardium, peritoneum, tunica vaginalis testis)1,2. For pleural mesothelioma, the median overall survival age ranges from 21 months (for Stage I) to 12 month (for Stage IV) disease3. In the 1970s, the incidence of mesothelioma cases started to increase, and it became evident that the occupational and environmental exposures to asbestos (occurring during 1930s–1970s) were associated with the increased incidence of this fatal disease4. Despite regulations aimed to ban the industrial use of asbestos by US Occupational Safety and Health Administration (OSHA) in 1970, data do not suggest a decline in the incidence of malignant mesothelioma in the U.S.5. However, the impact of these changes are difficult to assess due to the fact that mesothelioma is typically diagnosed decades after the initial asbestos exposure6. A recent multisite cohort investigation reported that the median time of diagnosis from the first environmental exposure was 38.4 years (IQR 31.3–45.4 years)7.\n\nAfter the pleura, the peritoneum is the second most frequent site of origin of mesothelioma. The epidemiological studies of peritoneal mesothelioma are complicated by the rarity of this disease, as well as by possible geographic and temporal variations in diagnostic practices8. While survival for patients with peritoneal mesothelioma is more favorable, with patients surviving up to 60 months9,10, limited number of papers explored factors affecting the survival of peritoneal mesothelioma.\n\nHowever, given the rarity of the disease, few databases have a sufficient number of cases and treatment data to make analysis of therapeutic options with statistical significance possible. NMVB is an especially valuable resource for mesothelioma research, as it includes populations residing in Pennsylvania and New York states (two of the top 5 states for mesothelioma-associated mortality)11. Previous SEER (Surveillance, Epidemiology and End Result Program) based studies exploring factors that influence mesothelioma did not include populations residing in Pennsylvania and New York12.\n\nPreviously published research of pleural mesothelioma suggested that histological type (epithelioid) and early stages were associated with improved survival with surgical treatment13. Other predictive factors explored in previously published literature including gender, advanced age, weight loss, chest pain, poor performance status, as well as low hemoglobin, leukocytosis, and thrombocytosis. It has been suggested that female patients with mesothelioma have better life expectancy as compared to male patients14.\n\nCurrently there are few therapeutic options, including surgery, chemotherapy, radiation therapy and a combination of these options that may significantly improve the overall survival from this deadly disease15. Considering the aggressive nature and poor prognosis associated with this disease, improving our existing knowledge regarding the biology of the disease and factors predictive of the efficacy of existing therapeutic options and treatment regiments for malignant mesothelioma is critical.\n\nIn this study, we analyzed malignant mesothelioma cases from the National Mesothelioma Virtual Bank (NMVB) to evaluate the effect of clinical, pathological, and epidemiological factors, and therapeutic options as determinants of overall survival. Thus our study adds geographic breadth to the existing mesothelioma research knowledge. Additionally, our dataset includes cases of peritoneal mesothelioma, which were not the focus of previous studies.\n\n\nMethods\n\nThis study is conducted under the Institutional Review Board (IRB) approval (IRB #0608194) of NMVB and approval from the principal investigator of NMVB to use the de-identified data from the resource.\n\nThe patient cohort for this study (n=888) is selected from the NMVB resource, which contains both pleural and peritoneal malignant mesothelioma cases. The NMVB database only records general treatment type including cancer directed surgery alone, surgery combined with chemotherapy, as well as surgery combined with chemotherapy and radiation. The specific type of treatment (such as exact surgery type of type of chemotherapy regimen used) is not recorded in the NMVB. NMVB enrolls patients from NMVB collaborative sites (New York University, University of Pennsylvania, University of Maryland, Roswell Park Cancer Institute and University of Pittsburgh Medical Center) in the north east region of country. Thus, there may be a selection bias with patients because there are few patients enrolled from the other regions of the country due to NMVB network coverage. In addition, NMVB has developed to collect mesothelioma biospecimens and data from prospectively consented retrospectively identified patients.\n\nDemographic, treatment, clinical and survival information of histologically confirmed pleural and peritoneal mesothelioma patients diagnosed between 1999 and 2017 were obtained from the NMVB database. Inclusion criteria included the following: confirmed diagnosis of malignant mesothelioma (limited to pleural and peritoneal presentation), presence of complete data on age, gender, race, asbestos exposure, smoking history, history of alcohol use, histological type, site of tumor, disease stage (for pleural presentation), vital status, and survival period. Exclusion criteria included the following: benign mesothelioma, and tumor site other than pleura and peritoneum. This investigation was limited to the most common histological subtypes of malignant mesothelioma including biphasic, epithelial or epithelioid, and sarcomatoid. The desmoplastic histology subtype is classified as sarcomatoid, and papillary mesothelioma as epithelial or epithelioid16,17. For the purpose of this study, the tumor anatomic site is classified into two main categories: pleura (which includes visceral/parietal pleura and lung, chest wall, ribs) and peritoneum (includes peritoneal cavity and organs involved). This analysis focused on 888 participants that met the inclusion criteria. Patient characteristics are presented in Table 1. Case selection flow is presented in Figure 1.\n\nWe have performed analysis of staging data to pleural mesothelioma cases that have surgical resections, there is no formal TNM staging system for peritoneal malignant mesothelioma. We converted the TNM staging of pleural mesothelioma into stage grouping as per College of American Pathology (CAP) protocol 2017 for pleural malignant mesothelioma. The metastatic disease status was defined as the tumor spread from the point of origin to the lymph node and other organs in the body.\n\n\nStatistical analyses\n\nWe included the following variables in the analysis: age, gender, race, smoking history, history of alcohol, asbestos exposure, site of tumor, histological type, treatment, staging and outcome variables including vital status and survival period. Duration of observation was defined as time (in months) between date of initial diagnosis until death (vital status = expired) or the date of last known contact for each participant. Smoking history was analyzed as a dichotomous variable (yes/no), where current, past and smoking for a brief period of time, were grouped as positive history of smoking (yes). The contribution of the three treatment types on mesothelioma survival rate is evaluated in this study.\n\nWe constructed survival curves using the Kaplan-Meier method for the entire dataset, followed by a separate analysis limited to female patients. We also performed a separate Kaplan-Meier analysis for peritoneal cases only. We performed Log-rank test of equality across strata for categorical variables. We analyzed the independent contribution to mesothelioma survival of several prognostic with univariable and multivariable regression methods based on the Cox proportional hazards model. Variables were entered into the model using a forward selection approach, starting with the most significant variable (based on the unadjusted p-value) and then continuing in order of significance. We analyzed factors contributing to mesothelioma survival separately for cases with complete data and with missing data to rule out any systematic bias associated with cases with missing data. Two-tailed p-values less than 0.05 were considered significant. We used The R Project (version 3.4.0) for Statistical Computing to perform all analysis18.\n\n\nResults\n\nThe majority of patients were European American (97%) and male (77%). Positive history of smoking has been reported by 364 (57 %) patients among n=641 and positive history of asbestos exposure has been reported by 413 cases (78 %) among n= 531. The epithelial or epithelioid histological subtype was the most prevalent histology in this dataset (n = 636), in 71.4% of cases. Cancer directed surgery has been performed in 54 % cases, while surgery and chemotherapy treatment jointly has been administered in 37% of cases. The median overall survival of the cohort was 15 months. Table 2 and Figure 3 demonstrate the results of the univariable and multivariable analysis respectively (Cox proportional hazard regression models).\n\nKaplan Meier Curve analysis performed at age (a), gender (b), anatomic site (c), histology subtype (d), history of asbestoses exposure (e), staging (pleural mesothelioma) (f), therapy type (g), and history of smoking (h).\n\nOverall, the non-parametric univariate Kaplan Meier analysis and log rank tests demonstrated longer survival in younger age group (18–44 years), female gender, with no known asbestos exposure history, epithelioid histological type, combined surgical and chemotherapy, Stage I, or peritoneum presentation (Figure 2a–2i).\n\nThe median survival for age group 18–44 years was 59 months (95% CI: 34 - 91) but much less favorable for the age group 75 and over, at 10 months (95% CI: 9 – 13). The median survival for females was 22 months (95% CI: 18 - 30) as compared to 14 months for males (95% CI: 13-16). The group with no reported history of asbestos exposure had a median survival rate of 20 months (95% CI: 16 - 31), as compared to median survival of 15 months (95% CI: 13-17) for the group with reported exposure. The epithelioid histological type median had a median survival of 18 months (95% CI: 17-21) as compared to 10 months for biphasic (95% CI: 9-13) and 7 months for sarcomatoid subtype (95%CI: 6-11). The European American group had a median survival of 15 months (95% CI: 13 – 16) as compare to median survival of 34 months (95% CI: 21-83) in non-European American population. The analysis suggests patients receiving combined therapies [(surgical and chemotherapy (95% CI: 13-19), surgical plus chemotherapy and radiation therapy (95% CI: 10-21)] had a more favorable median survival period in comparison to those with single line surgical therapy (95% CI: 8-14). Overall, median OS was most favorable (23 months (95% CI 21 to 27 months)) for patients treated with combined surgery and chemotherapy. Adding radiation to chemotherapy did not improve survival.\n\nThe median survival period for stage I group (including stages IA and IB) was 20 months (95% CI: 18 – 25) as compared to 12 months for stages III and IV. Presentation in the peritoneum site and no history of smoking was also associated with improved survival (Figure 1). When stratified by anatomic site of tumor, the median survival period among patients with peritoneal mesothelioma, who received surgical and chemotherapy, demonstrated longer survival of 28 months (95% CI: 28 – 45) as compared to 14 months (95% CI: 11 – 17) in patients with pleural mesothelioma.\n\n. Overall, multivariable analysis confirmed that younger age groups, female gender, peritoneal anatomic site, combination of surgery and chemotherapy, no history of smoking, early stage (I and II), and epithelial histology were all predictors of more favorable survival (Table 2).\n\nIn addition, we performed multivariable cox hazard proportional analysis on the complete dataset of n= 477 which had no missing record variables that has obtained from the primary dataset (n= 88). We included all the predictive prognostic variables except for stage, because there is no established TNM staging for peritoneal mesothelioma. We presented these results as supplementary analysis in Figure 3.\n\n\nDiscussion and conclusion\n\nThe focus of this study has been on the exploration of risk factors affecting mortality in the states of Pennsylvania and New York, a region with an aging population, environmental concerns, history of notable asbestos exposure, and other risk factors associated with mesothelioma development. This region has not been covered by previously reported investigations. In addition to expanding the geographic region in this study, another added value of this study is that we explored factors contributing to survival for peritoneal mesothelioma separately from the more prevalent pleural presentation. Survival analysis on the NMVB cohort demonstrated that being aged 44 and under, female gender, epithelioid histological subtype, Stage I of the disease, peritoneum anatomic site and surgical therapy combined with chemotherapy were favorable prognostic factors. This study corroborates the analysis of the SEER data by Taioli et al. suggesting that female gender, younger age, early stage, and surgery alone were all prognostic factors12. This study also corroborates previous investigations suggesting that peritoneal presentation, especially among women, is associated with longer survival19.\n\nConsistent with the literature, our data suggests that women have longer survival in comparison to men, which may be due to factors like lower levels of smoking amongst females and/or different levels of environmental exposures14,20–23. Specifically, women may be more likely to have para-occupational exposures, which typically refer to an asbestos-exposed worker serving as a vector for the transport of fibers to the household setting and family members. Other terms used in this context include household contact, take-home exposure or domestic exposure24. Exact factors explaining survival advantage among women needs to be further investigated in future research.\n\nStrengths of this study include the use of a very large dataset collected utilizing uniform data collection protocol. The weaknesses of this study include missing information on specific surgical treatment type in this dataset. Additionally, while we attempted to obtain detailed occupational exposure data for asbestos and other substances, participants’ ability to recall the duration and details of their exposure is a potential source of bias.\n\nMalignant mesothelioma is a life-threatening condition that has been under investigated and is important to investigate further, considering that its mortality epidemic has not shown signs of improvement in the past several decades. Further studies are needed to evaluate screening, diagnostic, staging and treatment for various subtypes of mesothelioma.\n\nIn the future, it would be particularly interesting to identify and evaluate cases of nonsurgical mesothelioma management because many patients are not good candidates for surgery. An improved understanding of factors associated with mesothelioma morbidity and mortality may help identify high-risk groups with different occupational exposures who should be further evaluated for responsiveness to preventive and innovative management strategies for mesothelioma. The identification of these factors could help patients at risk for therapy failure who may benefit from novel interventions or avoiding treatments that are not effective or with high mortality risk. We hope our report underscored the significant value of NMVB as a national research resource open to all research community and envision that in the future, existing information repositories like NMVB will be harnessed to greater extent to investigate rare diseases like mesothelioma.\n\n\nData availability\n\nThe investigator can obtain the de-identified data from National Mesothelioma Virtual Bank by submitting the letter of intent (LOI) (https://mesotissue.org/node/26). The NMVB Research Evaluation Panel (REP) is composed of extramural scientists with varied expertise including laboratory science, lung pathology, mesothelioma, and statistics (https://mesotissue.org/rep) reviews scientific merit of requests for NMVB specimens/data and makes recommendation to fulfil the request after the approval of data use agreement (DUA).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is funded and supported by the Centers for Disease Control and Prevention (CDC) in association with the National Institute for Occupational Safety and Health (NIOSH) Grant [5U24OH009077-11].\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\nLanphear BP, Buncher CR: Latent period for malignant mesothelioma of occupational origin. J Occup Med. 1992; 34(7): 718–21. PubMed Abstract\n\nSelikoff IJ, Hammond EC, Seidman H: Latency of asbestos disease among insulation workers in the United States and Canada. Cancer. 1980; 46(12): 2736–40. PubMed Abstract \| Publisher Full Text\n\nEiseman E, Bloom G, Brower J, et al.: Case studies of existing human tissue repositories: \"Best Practice\" for a biospecimen resource for the genomic and proteomic era. Santa Monica, CA: RAND; 2003. Reference Source\n\nYang H, Testa JR, Carbone M: Mesothelioma epidemiology, carcinogenesis, and pathogenesis. Curr Treat Options Oncol. 2008; 9(2–3): 147–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrice B, Ware A: Time trend of mesothelioma incidence in the United States and projection of future cases: an update based on SEER data for 1973 through 2005. Crit Rev Toxicol. 2009; 39(7): 576–88. PubMed Abstract \| Publisher Full Text\n\nBianchi C, Bianchi T: Global mesothelioma epidemic: Trend and features. Indian J Occup Environ Med. 2014; 18(2): 82–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReid A, de Klerk NH, Magnani C, et al.: Mesothelioma risk after 40 years since first exposure to asbestos: a pooled analysis. Thorax. 2014; 69(9): 843–50. PubMed Abstract \| Publisher Full Text\n\nBoffetta P: Epidemiology of peritoneal mesothelioma: a review. Ann Oncol. 2007; 18(6): 985–90. PubMed Abstract \| Publisher Full Text\n\nMohamed F, Sugarbaker PH: Peritoneal mesothelioma. Curr Treat Options Oncol. 2002; 3(5): 375–86. PubMed Abstract \| Publisher Full Text\n\nSebbag G, Yan H, Shmookler BM, et al.: Results of treatment of 33 patients with peritoneal mesothelioma. Br J Surg. 2000; 87(11): 1587–93. PubMed Abstract\n\nAmin W, Parwani AV, Schmandt L, et al.: National Mesothelioma Virtual Bank: a standard based biospecimen and clinical data resource to enhance translational research. BMC Cancer. 2008; 8: 236. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaioli E, Wolf AS, Camacho-Rivera M, et al.: Determinants of Survival in Malignant Pleural Mesothelioma: A Surveillance, Epidemiology, and End Results (SEER) Study of 14,228 Patients. PLoS One. 2015; 10(12): e0145039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyerhoff RR, Yang CF, Speicher PJ, et al.: Impact of mesothelioma histologic subtype on outcomes in the Surveillance, Epidemiology, and End Results database. J Surg Res. 2015; 196(1): 23–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdwards JG, Abrams KR, Leverment JN, et al.: Prognostic factors for malignant mesothelioma in 142 patients: validation of CALGB and EORTC prognostic scoring systems. Thorax. 2000; 55(9): 731–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiddinga BI, Rolfo C, van Meerbeeck JP: Mesothelioma treatment: Are we on target? A review. J Adv Res. 2015; 6(3): 319–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTravis WD: Sarcomatoid neoplasms of the lung and pleura. Arch Pathol Lab Med. 2010; 134(11): 1645–58. PubMed Abstract\n\nButnor KJ, Sporn TA, Hammar SP, et al.: Well-differentiated papillary mesothelioma. Am J Surg Pathol. 2001; 25(10): 1304–9. PubMed Abstract\n\nR Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2013. Reference Source\n\nSugarbaker PH, Welch LS, Mohamed F, et al.: A review of peritoneal mesothelioma at the Washington Cancer Institute. Surg Oncol Clin N Am. 2003; 12(3): 605–21, xi. PubMed Abstract\n\nMirabelli D, Roberti S, Gangemi M, et al.: Survival of peritoneal malignant mesothelioma in Italy: a population-based study. Int J Cancer. 2009; 124(1): 194–200. PubMed Abstract \| Publisher Full Text\n\nCurran D, Sahmoud T, Therasse P, et al.: Prognostic factors in patients with pleural mesothelioma: the European Organization for Research and Treatment of Cancer experience. J Clin Oncol. 1998; 16(1): 145–52. PubMed Abstract \| Publisher Full Text\n\nFlores RM, Zakowski M, Venkatraman E, et al.: Prognostic factors in the treatment of malignant pleural mesothelioma at a large tertiary referral center. J Thorac Oncol. 2007; 2(10): 957–65. PubMed Abstract \| Publisher Full Text\n\nLinton A, Pavlakis N, O'Connell R, et al.: Factors associated with survival in a large series of patients with malignant pleural mesothelioma in New South Wales. Br J Cancer. 2014; 111(9): 1860–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoonan CW: Environmental asbestos exposure and risk of mesothelioma. Ann Transl Med. 2017; 5(11): 234. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "37581", "date": "29 Aug 2018", "name": "Nico van Zandwijk", "expertise": [ "Reviewer Expertise Thoracic 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\n888 cases (out of 1400 cases) enrolled in the NMVB, representing around 1% of all mesothelioma cases (occurring from 1900 till 2107) in the US, are being used for this prognostic factors study.\nComparing the distribution of patients in this study with epidemiological studies suggests that over-representation of surgical and peritoneal cases may be present in the series presented. Multivariate analyses in a skewed population may give rise the wrong conclusions, and statistical/epidemiological advice is needed to assure that the conclusions from current analysis are valid.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4045", "date": "09 Oct 2018", "name": "Waqas Amin", "role": "Author Response", "response": "We would like to thank Reviewer 1 for their thoughtful comments. We would like to point out that, while our paper focuses on 1% of national mesothelioma cases, describing this population is extremely valuable as this group of patients is not a part of SEER and has not been captured by previous research. Findings should be considered in the context of related findings from other populations. While the role of aggressive surgery remains controversial for this groups of patients, few epidemiological studies have evaluated treatment patterns of these patients. The significance of these results, consistent with Reviewer’s comments, is further motivated by the fact that existing studies are flawed by their limited size and inclusion criteria. In the updated version of this paper, we commented (Discussion section) on selection of patients being a potential bias. We also commented on the fact that the treatment of our patients were consistent with ASCO guidelines. Also in the discussion, we pointed out that our conclusions are based on this particular group of patients and more extensive research needs to be implemented on the national and global level to draw more accurate conclusions.We also acknowledge regression analysis may be insufficient to control for confounding if groups are not largely overlapping. In the case of single exposures or assessing treatment effectiveness, causal inference methods (e.g. propensity score-based methods) may be more appropriate. One of our co-authors (Landsittel) is very familiar with these methods, having served as a PI of a methods contract on the topic (see https://www.pcori.org/research-results/2013/guidance-researchers-optimal-methods-conducting-comparative-effectiveness). However, we did not feel that these methods were entirely applicable since the goals of this project focused on describing a range of risk factor associations, which was still best accomplished through regression. Future analyses could focus on using such methods for more refined questions about a specific exposure.We have two coauthors, an epidemiologist (Linkov; associate professor of Ob/Gyn and Epidemiology) and a biostatistician (Landsittel; professor of biomedical informatics), who actively participated in the development of this paper, as well as data analysis. Their qualifications, that uniquely correspond to the primary focus of this research, are outlined below. Each has expertise in relevant methods, exposures and disease outcomes, has over 100 publications, and has 15-20 years of experience in research." } ] }, { "id": "38743", "date": "10 Oct 2018", "name": "Michele Carbone", "expertise": [ "Reviewer Expertise mesothelioma and asbestos", "environmental carcinogenesis", "gene environment interaction", "cancer syndromes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 done comprehensive report of analyses performed by a distinguished team of investigators who studied mesothelioma survival in the NMVB. The information presented is certainly useful to the scientific community. Most NMVB cases are from the States of New York and Pennsylvania where the Authors note there was significant exposure to asbestos.\n\nCritiques:\nMesotheliomas developing in carriers of germline mutations have significant improved survival (see Consensus Report Carbone M., Kanodia S., et al1).The lack of information about genetics is a limiting factor that should be acknowledge and that likely influences the finding that young age is a predictor of prolonged survival as these mesothelioma characteristically occur in young patients.In short this issue should be discussed. The information about asbestos exposure is based on self reported history.This information is often unreliable, as patients who think to have been exposed may not have been exposed and vice versa (asbestos is invisible by the naked eye so it is impossible to be certain whether dust contains or does not contain asbestos fibers, unless the dust is studied at the microscope), as shown for example by comparing results of lung content analyses and self reported history of exposure: see, Carbone M. et al2.The lack of corroborating evidence, such as radiological analyses supporting exposure –about 75% of patients exposed to asbestos develop bilateral plaques, should also be acknowledged. Most recent studies report that presently most pleural mesotheliomas occur in asbestos exposed individuals, and that instead patients with peritoneal mesothelioma rarely report asbestos exposure (for example only 5/64 patients in a recent series by Richard Alexander. Lee M et al3. (What was the proportion of self reported asbestos exposure among patients with pleural or peritoneal mesothelioma? Introduction. Mesothelioma is associated with exposure to professional exposure to asbestos and to environmental exposure to various mineral fibers including asbestos. Clarify this issue, and define what asbestos is. See Baumann F., Buck BJ, et al4; Baumann F et al5. Moreover, mesotheliomas develops in carriers of germline mutations of BAP1 (Carbone M., Kanodia S., JTO 20166, and mutations of BAP1 may increase susceptibility to low doses of asbestos and other mineral fibers (Napolitano A., Pellegrini L., et al7). These issues are important to understand the reasons of the current ongoing mesothelioma epidemic and also given the different prognosis and survival of mesotheliomas occurring in carriers of BAP1 mutations.\nMinor: Abstract conclusion last line…..treatment IN PERITONEAL MESOTHELIOMA is associated with improved survival….\n\nPage 3, introduction, bottom, therapeutic options: ref 15 in the rapidly evolving field of mesothelioma therapy is rather old. Replace or add current reference: the most recent review on this topic is Mutti L., Peikert T., et al8.\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": "4305", "date": "19 Dec 2018", "name": "Waqas Amin", "role": "Author Response", "response": "We have highlighted the importance mesotheliomas developing in carriers of germline mutations have significant improved survival. In the limitations, we recognize the fact that we do not have genetic information. We discuss the issue environmental exposure (Natural Occurring Asbestos) and occupational exposure and their association with pleural and peritoneal mesothelioma in introduction section. We recognize that we do not have radiological analysis to support exposure and rely on self-reported data. In our cohort of analysis, the ratio of pleural mesothelioma with asbestos exposure to malignant peritoneal mesothelioma with asbestos exposure is 23:277. Information on various exposures and BAP1 has been added to the introduction We have updated the citations in the manuscripts and made minor corrections." } ] }, { "id": "39375", "date": "19 Oct 2018", "name": "Tobias Peikert", "expertise": [ "Reviewer Expertise Pulmonary Medicine and Thoracic 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 manuscript summarizes the data from a large cohort of patients with pleural and peritoneal mesothelioma from the National Mesothelioma Virtual Tissue Bank (NMVB). The cases are from New York and Pennsylvania. The study confirms findings from prior analysis of the SEER database, which did not include patients from these states. Patient survival is dependent on age, gender, disease stage, disease site (pleural versus peritoneal), histological subtype and presence of multi-modality therapy. However there are several limitations.\nAs with most large mesothelioma databases surgically treated patients are over represented. In fact only 10-15% of patients with mesothelioma are candidates for surgery and most patients are treated with systemic chemotherapy. Consequently despite being a large cohort the study population is not representative of the majority of mesothelioma patients. Future studies should include a representative proportion of non-surgical patients. The conclusions about differences in therapy are difficult to interpret since not detailed treatment data (curative versus palliative), R1 versus >R1 resection, type of surgery and type of radiation therapy are not collected. It is also not clear of the patients in fact completed all therapies listed. In addition treatment data was only available in a subset of patients. In regards to the younger age patients BAP1 mutation status would be very helpful to explore. Some of this data should be available since the NMVB data set has been used for multiple correlative studies. It would be interesting to know if molecular analysis or immune staining are available for a subset of patients. The observation of a trend towards improved survival for non-Caucasian Americans is also very interesting and deserves further exploration.\n\nMinor comments:\n\nThe text on page 5 regarding the differences between the therapeutic groups should list the mean survival for the groups and not only the CI. (Also, could a single line of surgery been palliative pleurodesis?)\n\nPage 6 the n for the primary data set should be changed from 88 to 888.\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": "4304", "date": "19 Dec 2018", "name": "Waqas Amin", "role": "Author Response", "response": "We agree that our large cohort may not be representative of the general population of such patients and we recognize this weakness in the limitations section. NMVB cohort had very small number of cases that were not surgical candidates for their treatment of disease. We have not analyzed them because of insufficient number for meaningful statistical analysis. The specific treatment information has not collected in NMVB database. However, in future studies we will pull the information of specific treatments and include them in the analysis. Again, this limitation is recognized in the paper. We have highlight the issue related to younger age patients BAP1 mutation status in our introduction section paragraph 2. We would like to point out that we do not have such information in our database as of December 2018. There is a significant number of non-European American participants in the NMVB cohort due to geographic location of resource and its participants. We will further explore racial differences in survival in our future papers. Response to Minor Comments:Corrected." } ] } ]	1	https://f1000research.com/articles/7-1184
https://f1000research.com/articles/8-774/v1	03 Jun 19	{ "type": "Case Report", "title": "Case Report: Myiasis as a rare complication of invasive ductal carcinoma", "authors": [ "Muhammad Khurram Zia", "Syeda Ifra Asad", "Hafiz Abdul Wase", "Osama Salam", "Syed Zawahir Hassan", "Muhammad Musab", "Syed Mumtaz Ali Naqvi", "Hafiz Muhammad Furqan Izhar", "Muhammad Khurram Zia", "Syeda Ifra Asad", "Hafiz Abdul Wase", "Osama Salam", "Muhammad Musab", "Syed Mumtaz Ali Naqvi", "Hafiz Muhammad Furqan Izhar" ], "abstract": "Invasive ductal carcinoma (IDC) is the most common subtype of breast tumor. There were many cases reported about the treatment and adjuvant therapies. The simultaneously occurrence of breast carcinoma with cutaneous myiasis is, to our knowledge, a unique presentation. A 50-year-old female known case of breast cancer presented to the surgical department at Ziauddin Hospital Karachi with complaints of pain, redness, blackening, and a foul smelling, discharging wound on her left breast. The wound was debrided thoroughly with povidone-iodine and about 52 maggots were removed, which were identified as Chrysomya bezziana. The patient was hospitalized and received amoxicillin and ivermectin according to protocol. This case report is pertinent to public health professionals and oncologists in the view of the social impact of myiasis.", "keywords": [ "Myiasis", "Parasitic Infection", "Invasive ductal carcinoma" ], "content": "Introduction\n\nBreast carcinoma is the second-leading cause of cancer-related mortality in women. Invasive ductal carcinoma is most common histological subtype of breast carcinoma1. Myiasis is the dipterous larvae infestation of human or animal tissues. It is typically associated with inadequate personal hygiene due to lack of awareness among individuals in tropical and subtropical countries. Dipterous larvae in the feeding life cycle of may be found in living or dead tissues2–5. Many treatment options are available for invasive ductal carcinoma found with gangrenous tissues, with radiotherapy considered highly commendable in order to reduce disease recurrence6,7.\n\n\nCase report\n\nA 50-year-old female presented to the surgical department at Ziauddin Hospital Karachi with complaints of pain, redness, blackening, and a foul smelling and discharging wound in the left breast. She had a history of invasive ductal carcinoma which was diagnosed 2.5 years previously but could not get treatment for financial reasons. (Figure 1). On examination, the patient looked weak and lethargic. She had a fever of 101°F (38.3°C) The majority of the breast was hard in consistency with a purulent sanguineous discharging ulcer, which was foul smelling due to superimposed bacterial infection. The surrounding skin was gangrenous and numerous grayish maggots were seen crawling around.\n\nThe wound was debrided thoroughly with povidone-iodine in the emergency room and about 52 maggots were removed carefully, preserved and sent for entomological review which were identified as Chrysomya bezziana. The maggots were 15 to 20 mm long, whitish or greyish in color without body process. There is an enlargement of anterior spiracle and darkened portion of trunks, posterior spiracle extended three or four abdominal segments (Figure 2 and Figure 3). The patient was given wide-spectrum antibiotic amoxicillin 1 g twice a day for 7 days and Ivermectin 6 g twice a day for 1 day according to protocol8. Total mastectomy was performed in the oncology department at Ziauddin Hospital as part of palliative treatment due to the gangrene and myiasis. She was also started on radiotherapy and chemotherapy for ductal carcinoma; the chemotherapeutic regimen included capecitabine 1200 mg/m2 twice a day for 21 days with repeat cycles after every 21 days for 6 weeks. Total radiation dose was 5000 cGy delivered in 25 fractions 5 days a week for 6 weeks. Two months after ceasing her treatment with ivermectin and amoxicillin, breast tissue was healed and surrounded by scarring.\n\n\nDiscussion\n\nCutaneous myiasis is a rare entity. It is an infestation of human skin with maggots of flies which feed on host tissues. There are two classifications of myiasis, anatomical and ecological. The first description of myiasis was given by Hope in 1840. Since then many cases of myiasis affecting different human organs have been described2,3,6,7. Francesconi4 used an anatomical classification of myiasis, in which it is grouped into sanguinivorous or bloodsucking, cavitary, wound, cutaneous, furuncular and migratory myiasis.\n\nFlies lay eggs which hatch in a humid and warm environment, and larvae can get access directly to skin from wet clothes, buds or insects. Cutaneous myiasis is very uncommon; the majority of cases are caused by human botfly (Dermatobia hominis)5,8,9. Cutaneous myiasis is presented as a slow developing ulcer or boils. Some of the physical presentations of mastitis are similar to those of carcinoma of the breast10. It is important to note that an affected breast with myiasis, which appears like fungating mass with an ulcer, can be sometimes misdiagnosed and is confused with tuberculosis, mycosis, actinomycosis, furunculosis, chronic breast abscess, fungating malignancies, periductal mastitis, inflammatory carcinoma of the breast and cellulitis10,11. Therefore, it is very important to keep this rare but possible disease in the differentials when diagnosing the condition.\n\nSample larvae should be promptly preserved after removal in order to maintain their identity, because subsequent treatment is based on the type of the organism identified. Various techniques are available to remove larvae without affecting their shape and structure12,13. Risk factors should also be kept in mind, such as living in endemic areas, the characteristic intense itching of the affected breast, offending maggots (seen via a hand magnifying glass) are invaluable aids to the diagnosis and treatment. Poor personal hygiene is an important cause of myiasis;. it can be prevented by proper sanitation, good personal hygiene, spraying insecticides for flies and removal of garbage from nearby streets. Clothes should be worn after washing, drying in sunlight and ironing in order to prevent myiasis14.\n\nAfter the treatment of myiasis, any remaining ulcer should be biopsied in order to rule out any malignancy, as in our case, cutaneous (breast) myiasis simultaneously occurred with invasive ductal carcinoma (Figure 2) and after surgical removal (Figure 3) was confirmed with a biopsy (Figure 1). Invasive ductal carcinoma is the most common type of breast cancer, making up nearly 70–80% of all breast cancer cases. Invasive or infiltrative ductal carcinoma is the presence of abnormal cancer cells in the lactiferous ducts that have spread into other parts of the breast tissue. It can also metastasize to other parts of the body. Histological examination of breast cancer is mandatory to confirm the diagnosis and to establish different pathological prognostic factors15,16.\n\n\nConclusion\n\nCutaneous myiasis with breast cancer is a rare but possible entity and we should include it in our differentials which is important to avoid any further delay in diagnosis and adequate treatment in the future.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in funding this work.\n\n\nReferences\n\nEllis IO, Elston CW: Tumors of the breast. In: Fletcher CDM. (Ed.): Diagnostic Histopathology of Tumors. Churchill Livingstone, London, 1995.\n\nHope FW: On insects and their larvae occasionally found in the human body. Trans R Soc Entomol. 1840; 2: 256–271. Reference Source\n\nErfan F: Gingival myiasis caused by Diptera (Sarcophaga). Oral Surg Oral Med Oral Pathol. 1980; 49(2): 148–150. PubMed Abstract \| Publisher Full Text\n\nEdwards KM, Meredith TA, Hagler WS, et al.: Ophthalmomyiasis interna causing visual loss. Am J Ophthalmol. 1984; 97(5): 605–610. PubMed Abstract \| Publisher Full Text\n\nFerrar P: A guide to the breeding habits and immature stages of Diptera Cyclorrhapha. Entomonograph. 1987; 8: 907. Reference Source\n\nRudan N, Fajdi J, Stanec Z: Kirurgijadojke. Globus, Zagreb, 1998.\n\nSamija M, Krajina Z, Puru[I] A: Radioterapija. Nakladnizavod Globus, Zagreb, 1996.\n\nFrancesconi F, Lupi O: Myiasis. Clin Microbiol Rev. 2012; 25(1): 79–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang C: A collective analysis on 54 cases of human myiasis in China from 1995-2001. Chin Med J (Engl). 2002; 115(10): 1445–1447. PubMed Abstract\n\nde Barros N, D'Avila MS, de Pace Bauab S, et al.: Cutaneous myiasis of the breast: mammographic and US features-report of five cases. Radiology. 2001 ; 218(2): 517–520. PubMed Abstract \| Publisher Full Text\n\nFacina G, Nazario ACP, Kemp C, et al.: Myiasis by Dermatobia hominis mimicking periductal mastitis. Rev Bras Mastol. 1999; 9: 84–85.\n\nKahn DG: Myiasis secondary to Sermatobia hominis (human botfly) presenting as a long-standing breast mass. Arch Pathol Lab Med. 1999; 123(9): 829–831. PubMed Abstract\n\nUgwu BT, Nwadiaro PO: Cordylobia anthropophaga mastitis mimicking breast cancer: case report. East Afr Med J. 1999; 76(2): 115–6. PubMed Abstract\n\nKwong A, Yiu WK, Chow LW, et al.: Chrysomya bezziana: a rare infestation of the breast. Breast J. 2007; 13(3): 297–301. PubMed Abstract \| Publisher Full Text\n\nOlumide YM: Cutaneous myiasis: a simple and effective technique for extraction of Dermatobia hominis larvae. Int J dermatol. 1994; 33(2): 148–9. PubMed Abstract \| Publisher Full Text\n\nBoggild AK, Keystone JS, Kain KC: Furuncular myiasis: a simple and rapid method for extraction of intact Dermatobia hominis larvae. Clin Infect Dis. 2002; 35(3): 336–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56440", "date": "18 Nov 2019", "name": "Stefano Veraldi", "expertise": [ "Reviewer Expertise Infectious and parasitic diseaes of the skin. Tropical dermatology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of ulcerative breast carcinoma with Chrysomya bezziana infestation.\nIt is necessary to explain the reasons for which ivermectin was used. In addition: why this dosage and duration?\n\nIt is also necessary to add that Cordylobia anthropophaga is typical of Western Africa, Cordylobia rodhaini of Eastern Africa and Dermatologia hominis of Latin America.\n\nA language edit is required.\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": "77491", "date": "26 Jan 2021", "name": "Yunjiang Liu", "expertise": [ "Reviewer Expertise breast cancer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented a rare breast invasive ductal carcinoma complicated with cutanous Myiasis.\n\nCan the authors provide more detail about physical examination and diagnostic tests of breast cancer? For example: did the authors performed CT scanning of liver, lung, bone and brain? They should present the disease stage of the case. Did they finish the immunohistochemistry staining of breast cancer, including hormone receptor (ER, PR) and cerbB-2? It will be useful for other practitioners.\nI’d like to remind the importance of breast cancer screening in developing countries.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-774
https://f1000research.com/articles/7-1833/v1	21 Nov 18	{ "type": "Research Article", "title": "Financing Agriculture in Nigeria through Agricultural Extension Services of Agricultural Development Programmes (ADPs)", "authors": [ "Henry Inegbedion", "Eseosa Obadiaru", "Barnabas Obasaju", "Abiola Asaleye", "Adedoyin Lawal", "Eseosa Obadiaru", "Barnabas Obasaju", "Abiola Asaleye", "Adedoyin Lawal" ], "abstract": "The ADPs were designed in response to a fall in agricultural productivity and hence a concern to sustain domestic food supplies. The study examined “Financing Agriculture in Nigeria through Agricultural Extension Services of Agricultural Development Programmes.” It sought to ascertain the extent to which agricultural extension services of the agricultural development programmes have impacted the financing of agriculture in six selected local government areas in Edo South senatorial district, Nigeria using a sample of 120 respondents. Stratified random sampling was used to select the respondents. Interview schedule served as the research instrument. The research data were analyzed using t-test and Pearson correlation, which served as the inferential statistics. The research findings showed that the extension services of ADP have impacted significantly on crop development in the selected communities but have not had significant impact on employment creation and the development of infrastructural facilities. The study also revealed that there was no significant difference between the implementation of the projects in the selected communities, as revealed by the correlation test. On the basis of the research findings, the need for a complete redesign of the project to ensure that it achieves its stated goals as well as ensure proper monitoring of its implementation were suggested, among others.", "keywords": [ "Agricultural Extension Services", "ADP", "Agricultural development" ], "content": "Introduction\n\nPrior to independence, agricultural production was the mainstay of the Nigerian economy. Even following independence the scenario did not show any change as agriculture contributed well over 90 percent to the nation’ foreign exchange earnings. However, with the discovery, exploitation, and exportation of crude oil in commercial quantities, the contribution of agriculture to the nation’s foreign exchange and GDP began to dwindle as attention shifted from agriculture to oil (Inegbedion, 2012). The domination of the economy by the oil sector caused the Nigerian economy to become a monoculture, with the attendant implications. This led to the disengagement of many able-bodied men from productive activities in search for oil money, thus precipitating an unprecedented rural-urban drift in the 1970s. The neglect of the agricultural sector and the subsequent rural-urban drift created a visible dent in Nigeria’s food supply, and thus signaled the need to embark on massive importation of food stuffs in the early 1980s.\n\nThe government’s response to the glaring distortion was to embark on a series of interventionist programmes referred to as the Agricultural Development Projects (ADPs). The ADPs were designed to increase crop production with the major components being improving technology, increasing the supply of farming and crop inputs, as well as reasonable improvements in basic infrastructure (Ammani et al., 2010). The ADP was kick started with three pilot programmes in the northern part of the country. The perceived success of the pilot programmes necessitated a clarion call for its expansion (Independent Evaluation Group, 2009: 1). This led to the replication of the projects from the early 1980s all through the decade. Bendel ADP, which later became Edo ADP in 1992, was among this group. Each of these groups included four basic components:- farm and crop development (expanded research, extension, and input supply), infrastructure development (feeder road construction and maintenance, water supplies, markets and storage facilities), institutional support, establishment of project entities separate from the state agriculture departments, and technical assistance, largely to manage the new institutions. Owing to the perceived importance of ADP to agricultural development in Nigeria several studies have been conducted to evaluate its performance since inception, some of the recent studies include and Adamu & Mohammed (2009); Ammani et al. (2010); Enwelu et al. (2017); Naswem & Ejembi (2017); Nchuchuwe & Adejuwon (2012); Omonijo et al. (2014); Ugwu (2007); Umeh et al. (2015), as well as Olujenyo (2006), among others.\n\nThe study sought, mainly, to investigate the impact of agricultural extension services of Edo state ADP on agricultural financing in Edo state using six selected communities in three local government areas in Edo south senatorial district, by focusing on the activities of Edo ADP Extension Division. The specific objectives of the study were to determine: The extent to which the extension services of Edo ADP have impacted farm and crop development in the selected communities under study; the extent to which the extension services of Edo ADP have impacted infrastructural development in the communities under its coverage, and the extent to which the extension services of Edo ADP have contributed to the reduction of unemployment through the attraction of able-bodied men and women to agriculture.\n\n\nLiterature review\n\nThere are numerous accounts of some countries that use to be buoyant but later nosedived, due partly to mismanagement of environmental resources. Learning from such accounts it is imperative that policy makers be conscious on the need for effective management of their environments with a view to minimizing the cost of producing natural products like food, fibers, and associated resources, while mindful of the need to minimize the risk of to the survival of future generations (Tunji Titilola, 2001) As a major component of economic development, the ADP was meant to boost production and productivity for some reasonable period of time as well as the wellbeing of farmers; which was supposed to translate to a higher per capital income of the economy. Rural development, which is part of the central focus of agricultural productivity, is not only concerned with a sustainable increase in the levels of productivity and production of farmers and other rural dwellers; neither is it only concerned with a significant enhancement of the wellbeing of rural dwellers as reflected in increased per capita income and standard of living, but it is also expected to translate to a substantial and sustainable enhancement in the social and economic wellbeing of the rural communities (Nchuchuwe & Adejuwon, 2012). Rural development can thus be seen as instrumental in combating deprivation and poverty in order to enhance economic prosperity at the grassroots. From the point of view of most countries, rural development refers to a sustainable increase in the productivity and earnings of households and low income workers from rural areas (Nwachukwu & Eze, 2007).\n\nDwindling agricultural productivity in the mid-70s necessitated the establishment of the ADPs; the goal was to sustain domestic food supplies in the country given the migration of labour from agriculture to other sectors that were perceived to be more lucrative at the time, especially in the cities, owing to the activities from the oil boom (Independent Evaluation Group, 2009. Available from the World Bank Group website). On the other hand, government was afforded the resources and opportunity to develop the ADPs by the domestic recycling of oil income. The ADPs, consistent with their purpose, provided opportunity for investment in agriculture and agricultural services, and infrastructural facilities such as access roads and pipe borne water in the rural areas. The establishment and adoption of the ADPs by government situated the smallholder sector at the center of its agricultural development strategy; this action marked a conspicuous shift from the previous capital intensive investment projects targeted at selected areas designated as having high agricultural potential.\n\nThe pioneer ADPs were community projects with each encompassing specific regions within a state. The results of the implementations were impressive to the policy makers at the federal, state and local governments. To this end, the governments were pressured to replicate the projects in all the remaining communities across all the states of the federation. As a result of the pressure and expectations, all 19 states of the federation had ADPs by 1989. The major goal was to increase food production and farm incomes in all host communities across the federation. The thinking was that increases in production and productivity would be a direct consequence of improved technology, especially planting material and fertilizer. The design of the agricultural components of the ADPs was centered on systems for the development and transfer of technology to farmers, and the distribution of modern inputs and land development. These included land clearing and small scale irrigation of irrigable areas in the northern parts of the country. Investments in infrastructural component of the programme included the construction of expanded feeder road network, construction of farm service centers for the distribution of crop and farm inputs, as well as the provision of facilities for the operations of ADP staff. With the exception of the Ilorin ADP, all the other projects across the country supported improvements in rural water supplies.\n\nEdo state ADP. ADP was established in Benin City in 1986 with focus on all the local government areas in the old Bendel state. With the creation of Edo state in 1991, the focus (coverage areas) of the scheme became restricted to all the local government areas in Edo state. The scheme is a tripartite arrangement between the federal, states, and local governments just like the programmes in northern Nigeria. That notwithstanding, at the moment, the state government is solely responsible for the payment of staff salaries. The areas of intervention of Edo ADP include; infrastructural development, such as construction and maintenance of earth roads,; provision of water through the sinking of Boreholes in the communities,; building of markets,; and provision of storage facilities,; as well as farm and crop development (rural agriculture) through the provision of fertilizers, pesticides and farm implements to rural farmers at subsidized rates as well as the provision of consultancy services and sensitization of farmers.\n\nThe FADAMA programme of the ADP, which began in the north has also taken off in the south, and is currently undertaken by Edo ADP. FADAMA means irrigable, and it involves interventionist programmes using relevant methodologies in areas were water is close to the surface of the earth. The frequency of intervention is contingent upon necessity as well as availability of funds, but also subject to political consideration at times. The ADP mandate includes farm and crop development, infrastructural development, institutional support and training, as well as consultancies. The major constraints currently being faced by Edo ADP include: Inadequate manpower, and the preference of the state ministry of agriculture over ADP by the administration of Edo state. The situation was alleged to be threatening the capacity of the Edo ADP to embark on its traditional interventionist programmes since such functions are now being contracted to the Ministry of Agriculture as of mid-2000. A framework of the study is presented (see Figure 1).\n\nNaswem & Ejembi (2017) investigated “reviving agricultural extension for effective transition from subsistence to commercial agriculture”. The purpose of their study was to identify the factors responsible for the erosion of the extension system, and identify a reliable path that will make the system come alive again. This was to trigger the new transformation agenda policy in agriculture. They highlighted the weaknesses of past extension efforts. The need for the younger generation to be deliberately involved in agriculture was suggested, among other recommendations.\n\nAmmani et al. (2010) investigated the “challenges to the sustainability of the ADP system in Nigeria”. The purpose of their study was to analyse the problems perceived to be constraining the sustainability of the ADP, and as a consequence, the effective performance of the ADP system in Nigeria. Inadequate funding was viewed as the focal problem. They developed and transposed a problem tree and used it to transform the identified root causes, and consequences into root solutions. Based on their findings, they suggested that government should focus on improving funding for the ADPs, making deductions from state and federal government revenue allocations from source through a counter-part funding arrangement for the ADPs.\n\nOlujenyo (2006) investigated the “impact of ADP on the quality of social existence of rural dwellers in developing economies in Ondo state (Nigeria)”. The purpose of the study was to examine the extent to which the implementation of the ADP had impacted the rural farmers in Ondo state of Nigeria, West Africa through an investigation of the impact of the programme on the farming operations of the target farmers, as well as the organizational, and farm-related factors perceived to be associated with the impact of the ADP. A survey design was employed and structured questionnaires served as the research instrument. The research instrument was used to ascertain the perception of 288 respondents about the performance of the ADP programme in terms of its impact on the rural farmers. The respondents consisted of 144 contact farmers and 144 non-contact farmers. Contact farmers are those who belong to cooperative societies, while non-contact farmers do not belong. Random and systematic sampling served as the sampling techniques. Research data were analysed using correlation technique. Following the inferential analysis of the data, it was found that average yields per hectare of land cultivated by the farmers differed significantly from the average score of the articles of convenience owned by the farmers before implementation and after the implementation of the ADP in all the four crops examined.\n\nNchuchuwe & Adejuwon (2012) sought to investigate the constraints to agricultural and rural development in Nigeria. They observe that agricultural activities in Africa were still largely traditional and mainly concentrated in the hands of pastoralists and smallholders and that the relegation of the agricultural sector has precipitated negative net migration. They further discuss the consequences occasioned by the problems and challenges resulting from the neglect of the agricultural sector, and government responses to the rural infrastructural needs of the people. Among other needs, they suggested the provision of an adequate level of strategically targeted investment in agriculture as well as the upgrade of rural infrastructure; concerted efforts at boosting of productivity and increased competitiveness of the farm output.\n\nUgwu (2007) examined “contributions of ADPs to rural livelihood and food security in Nigeria” He explored the programme from its inception in order to give a valuable account of its impact. It was observed that the primary purpose of establishing the ADPs was to stimulate agricultural production and thus help to bring about the enhancement of the wellbeing of the rural dwellers and provision of adequate food in all the benefiting communities in particular and the entire country in general. He identified the contributions of the ADPs to be mainly in the resuscitation of the extension service, enhancement of the knowledge of the local farmers, provision of basic infrastructure in the rural areas, provision of agricultural input to farmers,; development, transfer and adoption of adequate technology, as well as enhancement of the livelihood of individuals in the rural areas, and provision of adequate food as well. Furthermore, they observed that the continuity of the programme was guaranteed by the conspicuous successes of ADP in the target areas. However, political interference which are often unwarranted, unstable inflow of the required money due mainly to default in the payment of counterpart funding by the three tiers of government, rapid staff turn-over in most ADPs, among other factors, were identified as the major constraints to the success of the programme. The need for government to give increased political support was suggested.\n\nAdamu & Mohammed (2009) investigated “the effect of ADP on the rural farmers in Adamawa state, Nigeria”. The authors collected data on annual crop output, income, farm size, and the availability of improved technology, access to credit and training of farmers and rural infrastructure using a structured questionnaire and personal interviews,; t tests were used to analyze the data. The results indicated a positive, and significant impact of the ADP in Adamawa state on the productivity, income, access to credit, and standard of living of rural farmers using assets ownership criterion. The study did not reveal any significant impact of the ADP on the adoption of improved technologies, rural infrastructure, and farm sizes. Consequently, the need to enhance the provision of rural infrastructure, and technologies; as well as fund the project adequately was recommended, among others.\n\nOmonijo et al. (2014) examined “impacts of ADP on rural dwellers in Nigeria using the people of Isan Ekiti, Oye Local Government Area of Ekiti state as case”. A survey method was employed and a questionnaire served as the instrument. They retrieved and analysed 73 questionnaires using descriptive and inferential statistics. Multiple linear regression analysis served as the inferential test. Results reveal that there was a significant relationship between ADP (through increased provision of pesticides, improved seeds to farmers, establishment of new infrastructure, as well as provision of fertilizers) and increased food production in the locality. However, accessibility of credit by farmers had no significant effect on increased agricultural productivity. The need for government to increase its effort in the area of agricultural credit financing was suggested.\n\nAnother investigative study was conducted by Umeh et al. (2015). The study compared the performance of the ADP of Abia with that of Enugu states in Nigeria. The authors evaluated the performance of the programmes in the two states with particular focus on agricultural extension delivery services. They used multi-stage sampling to select 200 respondents. Primary and secondary data were employed. The paired t-test was used in hypothesis testing. Results showed that three out of the 11 performance indices of the two States were significantly different at the 95% level of confidence. Consequently, the need for government to expedite action in the employment of better trained extension staff to facilitate the enhancement of service delivery in Enugu State was suggested.\n\nEnwelu et al. (2017) investigated the “access and use of information communication technologies in Anambra State ADP”. A sample of 69 respondents was selected and investigated while a structured questionnaire served as the research instrument that was used to elicit the desired data. The data were analysed using descriptive and inferential statistics. The inferential statistics consisted of factor and regression analysis. The extension workers and workers in ADP office had high levels of access to mobile phones, and used phones very often. The major constraints to the access and use of other ICT facilities were found to be soaring cost of maintenance of ICT tools, dearth of competence in utilization of ICT and inadequate support by organization and government, among others.\n\n\nMethods\n\nThe study adopted conclusive research design in order to determine the extent to which the Agricultural Extension Services influences community development in Edo State. This is consistent with Inegbedion & Obadiaru (2018). The population of the study consisted of small scale farmers in Edo South senatorial district since they were the group to whom the generalization of the results of the study was intended (Agbonifoh & Yomere, 2002). Six communities, two each from three local government areas in the old Oredo local government area – Ikpoba-Okha, Egor, and Oredo local government areas were the focus of the study. The communities were Iyekogba, Ogba, Etete, Evboriaria, Egor and Utoka. The study was conducted between December 2012 and January 2013.\n\nThe sample size was computed using the formula n=z2p(1−p)e2,. Here, n represents the sample size, Z is the score corresponding to the level of significance and representing the abscissa on a normal curve in area α to minus infinity at the tails, while 1 – α is the confidence level corresponding to the level of significance, α , e.g., 95% for α = 0.05; e = α is the assumed level of precision, p is the estimated proportion of farmers in the population and q is 1-p. The value for Z is obtained from a normal distribution table, based on the area under the normal curve (Cochran, 1963; and Agbadudu, 2007).\n\nThe aim was to be 95% confident that the perception of the sampled respondents will not differ by more than five percent from the perception of the true population of the study. Furthermore, approximately 40% of the people in the communities are farmers. Consequently, n=z2x 0.4 x 0.60.052 = 368.8. This value was approximated to 366, hence a sample size of 366 was used. Given the sample size of 366, 61 respondents were scheduled to be sampled in each community. The study was done in the communities but the community centers around the village heads served as the take-off points in each of the communities. The communities were stratified according to the nearest 50 compounds to the residence of the village head. Subsequently, simple random sampling was used to select the desired number of respondents from each community. Thus, the sampling technique used was stratified random. An interview schedule was then used to elicit the necessary information from the sampled respondents. The choice of the interview schedule as the research instrument was informed by its flexibility which allows items to be adapted to the respondents’ level of education. The study was conducted between December 2012 and January 2013 in Edo state of South-South Nigeria by this author and a research assistant who was paid to assist in conducting the interview. The interview schedule had two parts – the Bio-data, which featured items on the respondents’ demographic characteristics and the core-subject matter, which featured items that addressed the research problem. The question-response format of the research questions was the Likert type five point scale with options ranging from a region of strong agreement (strongly Agree), through a neutral zone (Not Sure), to a region of strong disagreement (Strongly Disagree). As it is common with all likert-scale items, the questions sought to ascertain respondents’ perception of the research problem. The questionnaire used is available as appendix in this paper.\n\nResearch data were analyzed, using descriptive statistics such as mean, standard deviation, mean difference and standard error mean; as well as one sample t test and F ratio test (ANOVA) and regression analysis, which served as inferential statistics. The one sample t test was used to test for significance of the independent variables while regression was used to examine their predictive power. ANOVA was performed to determine the existence of differences in in ADP across locations. This is consistent with Inegbedion et al. (2016). Data analysis implemented using the Statistical Package for Social Sciences (SPSS) 20.0. The study was reported using the SRQR reporting guidelines.\n\n\nEthical considerations and consent\n\nThe study was an Assignment which formed part of the course work in Economic Analysis, in partial fulfillment of the requirements for the award of a Doctor of Philosophy (Ph. D) in Business Administration of the University of Benin, Nigeria. The ethics committee indicated no ethical approval was required for this study.\n\nCo-authors were included to assist in enhancing the quality of the work. Verbal consent of the respondents was sought after assuring them of their anonymity. The respondents thus gave their consent willingly as they fully understood the purpose of the study. Due to time restrictions and the low risk nature of the study, verbal and not written informed consent was obtained from participants.\n\n\nResults\n\nOut of the 366 respondents scheduled to be sampled, information was elicited from 248 of them. The age distribution of the respondents shows that 12 were under 30 years, 75 were in the age group 31–40 years, 105 were in the age group 41–50 years and 56 were above 50 years, thus indicating that majority of them were in the age group 41–50 years. The sex distribution shows that 169 of them were male while 79 of them were female, thus indicating that majority of the respondents were male. The distribution by marital status shows that 191 of them are married, 38 are single, 15 are widowed, while 4 are divorced; thus showing that majority of them are married. The distribution by highest educational level showed that 113 of them have Secondary School Certificate (SSCE), 82 had National Diploma or National Certificate Examination Certificate (ND/NCE); lastly, the distribution of the respondents by local government showed that 82 of them were from Oredo local government, 90 from Egor local government and 76 from Ikpoba Okha local government; thus majority of the respondents were from Egor local government area. The results of the data analysis are presented in Table 1–Table 6.\n\nSource: SPSS output\n\nSource: SPSS output\n\nSource: SPSS output\n\nAgric. Ext. Services: Agricultural extension services\n\nCrop Devpt: Crop development\n\nInfrast. Devpt: Infrastructural Development\n\nReduct. in Unemp: Reduction in unemployment\n\nAgric. Ext. Services: Agricultural extension services\n\nAgric. Ext. Services: Agricultural extension services\n\nSix hypotheses were tested. The results of the tests are presented below\n\nAgricultural Extension Services of Edo ADP vs. crop development. A comparison of Agricultural Extension Services with crop development in the communities revealed that the mean score associated with respondents perception of the extent to which Edo ADP has influenced crop development projects in their communities was 3.153 with a standard deviation of 1.08668 and a standard error mean of 0.069. When this was compared with a test value of 3, a mean difference of 0.1532 was observed. A t-test for equality of means revealed that this difference was significant at the five per cent level since the significant (2-tailed) probability of 0.027 is less than 0.05, the assumed level of significance. Consequently, at the 95% confidence level, the agricultural extension services of Edo ADP have significantly affected farm development in the communities (see Table 1).\n\nAgricultural Extension Services of Edo ADP vs. farm development. A comparison of Agricultural Extension Services of Edo ADP with farm development in the communities revealed that the mean score associated with respondents perception of the extent to which Edo ADP has influenced farm development projects in their communities was 3.165 with a standard deviation of 1.0689 and a standard error mean of 0.0679. When this was compared with a test value of 3, it resulted in a mean difference of 0.1653. A t-test for equality of means revealed that the resultant mean difference was significant at the five per cent level since the significant (2-tailed) P Value of 0.016 is less than 0.05, the assumed level of significance. Consequently, at the 95% confidence level, it is safe to conclude that the agricultural extension services of Edo ADP have significantly affected farm development in the communities (see Table 2).\n\nAgricultural Extension Services of Edo ADP vs. development of infrastructural facilities. A comparison of Agricultural Extension Services of Edo ADP with the development of infrastructural facilities in the communities revealed that the mean score associated with respondents perception of the extent to which Edo ADP has influenced the development of infrastructural facilities in their communities was 3.0618 with a standard deviation of 1.0179 and a standard error mean of 0.06464. When this was compared with a test value of 3, it resulted in a mean difference of 0.1653. A t-test for equality of means revealed that the resultant mean difference was not significant at the five per cent level since the significant (2-tailed) P-value of 0.340 was not less than 0.05, the assumed level of significance. Consequently, at the 95% confidence level, the agricultural extension services of Edo ADP have not significantly influenced the development of infrastructural facilities in the communities (see Table 3).\n\nA comparison of Agricultural Extension Services with reduction in unemployment revealed that the mean score associated with respondents’ perception of the extent to which Edo ADP has influenced the reduction of unemployment in their communities was 3.0504 with a standard deviation of 1.037 and a standard error mean of 0.06585. When this was compared with a test value of 3, it resulted in a mean difference of 0.1653. A t-test for equality of means revealed that the resultant mean difference was not significant at the five per cent level since the significant p value of 0.445 is not less than 0.05 the assumed level of significance. Consequently, at the 95% confidence level, the agricultural extension services of Edo ADP have not significantly influenced the reduction in the level of unemployment in the communities (see Table 4).\n\nRespondents’ perception vs. location. A comparison of respondents’ perception with their location revealed that there was a significant difference between respondents’ perception and location since the computed F and associated significant probabilities were 7.451 and 0.015 (p < 0.05) respectively, thus indicating that at 99% confidence level, the effect of ADP is different across the locations (see Table 5). A post hoc test revealed that the impact of ADP in Egor local government was not significantly different from Ikpoba-Okha but significantly different from Oredo (see Table 5 and Table 5.1).\n\nExtension services of Edo ADP vs. crop/farm and infrastructural development as well as reduction in unemployment. Table 6 presents the regression analysis with extension services of ADP serving as the dependent variable while crop/farm development, infrastructural development and reduction in unemployment are the independent variables. The intention of this model is to establish the nature of the relationship between extension services of ADP and the explanatory variables. Here, the argument is based on the fact that if Y is directly proportional to X, then X is also directly proportional to Y. the results show that the adjusted R square is 0.526, thus indicating that the independent variables explain 52.6% of the variation in the dependent variable. Furthermore, crop/farm development and infrastructural development were significantly related to extension services of ADP. The reduction in unemployment was not significant. The ANOVA results show a calculated F statistic of 9.2125, with an asymptotic significant probability of 0.00, thus indicating that the test is significant at the one per cent level. Thus, at the 95% confidence level, we can conclude that the overall significance of the model is good.\n\nThe findings show that agricultural extension services of ADP have had significant impact on crop and farm development. However, the results do not indicate significant impact on reduction in unemployment and development of infrastructural facilities in the sampled communities. The results are consistent with the findings of Olujenyo (2006); Omonijo et al. (2014); Ugwu (2007) and Umeh et al. (2015). Also, the non-significance of agricultural extension services of ADP on infrastructural facilities is consistent with the findings of Adamu & Mohammed (2009). Furthermore, the results show that the impact of ADP on the local governments is not the same. In other words, the impact of the ADP is more in some locations than others. This is consistent with Enwelu et al. (2017). Lastly, the regression results show that the development of infrastructural facilities is significantly related to agricultural extension services of the ADP but the t test for significance shows that agricultural extension services of the ADP has not had a significant impact on the development of infrastructural facilities. This may not be unconnected with the uneven impact of the ADP in the respective locations. This partial impact of ADP on the development of infrastructural facilities is a point of departure from previous studies that examined impact of ADP and declared the project non-significant without ascertaining the degree of non-significance of the project.\n\nThe proposed model shows that the agricultural extension programmes (institutional support and training and consultancies) of the ADP have been impactful in farm and crop development and so should be consolidated in these areas. But the programmes have not been impactful in infrastructural development and employment creation. If these goals are to be achieved, the programme should be repackaged (see Figure 2)\n\n\nConclusions\n\nThe agricultural extension services of ADP have so far impacted crop and farm development significantly, but have not had significant impact on infrastructural development and reduction in rural unemployment. The non-significance of the ADP on development of infrastructural facilities is due to the uneven implementation of the programme across locations as revealed by the ANOVA test. This accounts for the conflict between the results of the t test and the least squares test. To this end, the extension services have been impactful but have not fully exerted the desired impact on the communities in Edo state. The pattern of intervention in some of the areas was significantly different others.\n\nThis study has made significant contribution to agricultural research literature by updating previous studies on ADP in Nigeria. Furthermore, the study revealed that while crop and farm development was significantly impacted by the ADP across all locations, development of infrastructural facilities was impacted in some locations but not in others. The partial impact of ADP on the development of infrastructural facilities as a result of uneven implementation of the programme across locations is a major contribution of this study. The study also proposed a model to capture the impact of ADP on agricultural financing till date.\n\nThe study was not without limitations, which could constrain the generalizability of the results of this study. First, the inability of the researcher to obtain a completely random sample, owing to logistic concerns, especially transportation, in the rural areas was a limitation because randomness is a prerequisite for representativeness. Nevertheless, efforts were made to minimize this constraint through the use of alternative transport mediums like tricycles and motorcycles. Secondly, the reluctance of some sampled respondents to volunteer the desired information was a big challenge and could have further distorted the desired randomness in sample selection. The reluctance was tackled by adequate sensitization which eventually saw approximately 68% of the sample responding to the interview.\n\n\nRecommendations/suggestions for further studies\n\nIn view of the findings, the following recommendations are suggested: Firstly, concerned authorities should show adequate commitment and willingness to the success of the ADP by re-strategizing and reengineering the extension services with a view to making it more encompassing in order to yield the desired impact; secondly, concerted efforts should be made to use the ADP as a means of rejuvenating the agricultural sector to boost the self-reliance and self-sufficiency of the food production agenda of the federal government of Nigeria as well as the diversification of the nation’s resource base; lastly, there is the need to also redesign the scheme to address the shortcomings, which are currently threatening to truncate the programme. Future studies should attempt to increase the sample size used in this study in order to enhance the reliability of the findings consistent with the central limit theory.\n\n\nData availability\n\nF1000Research: Dataset 1. Participant responses from the Agricultural Development Project survey, https://doi.org/10.5256/f1000research.16568.d225570 Inegbedion et al. (2018).", "appendix": "Grant information\n\nThis work was supported by Landmark University, Nigeria.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Study questionnaire\n\nClick here to access the data\n\n\nReferences\n\nAdamu MU, Mohammed WA: The effect of agricultural development project (ADP) on the rural farmers in Adamawa state, Nigeria. Asian J Agric Rural Dev. 2009; 2(3): 405–410. Reference Source\n\nAgbadudu AB: Statistics for business and the social sciences, Benin: Uri publishing company limited. 2007.\n\nAgbonifoh BA, Yomere GO: Research methodology in the social sciences and Education, Benin City: Centrepiece Consultancy limited. 2002.\n\nAmmani AA, Auta SJ, Aliyu JA: Challenges to sustainability: Applying the problem tree analysis methodology to the ADP system in Nigeria. J Agric Ext. 2010; 14(2): 36–46. Publisher Full Text\n\nCochran WG: Sampling techniques. New York: John Wiley and Sons, Inc. 1963. Reference Source\n\nEnwelu IA, Enwereuzor SO, Asadu AA, et al.: Access and Use of Information and Communication Technologies by Extension Workers in Anambra State Agricultural Development Programme, Nigeria. J Agric Ext. 2017; 21(2): 152–161. Publisher Full Text\n\nIndependent Evaluation Group: Agricultural developments in Nigeria. 2009. Reference Source\n\nInegbedion HE: Oil price hike and the Nigerian economy, Saarbrucken, Germany: Lap Lambert Academic Publishing. GmbH & Co. KG. ISBN-10: 3659117986. 2012.\n\nInegbedion HE, Obadiaru ED: Modelling brand loyalty in the Nigerian telecommunications industry. Journal of Strategic Marketing. 2018. 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, Obadiaru E, Obasaju B, et al.: Dataset 1 in: Financing Agriculture in Nigeria through Agricultural Extension Services of Agricultural Development Programmes(ADPs). F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16568.d225570\n\nNaswem AA, Ejembi SA: Reviving agricultural extension for effective transition from subsistence to commercial agriculture in Nigeria. J Rural Soc Sci. 2017; 32(1): 3–20. Reference Source\n\nNchuchuwe FF, Adejuwon KD: The Challenges of agriculture and rural development in Africa: The case of Nigeria. International Journal of Academic Research in Progressive Education and Development. 2012; 1(3): 45–61. Reference Source\n\nNwachukwu IN, Eze CI: Impact of selected rural development programmes on poverty alleviation in Ikuano Local Government Area, Abia state Nigeria. African Journal of Food, Agriculture, Nutrition and Development. Kenya. 2007; 7(5): 1–17. Reference Source\n\nOlujenyo FO: Impact of Agricultural Development Programme (ADP) on the Quality of Social Existence of Rural Dwellers in Developing Economies The Ondo State (Nigeria) Agricultural Development Programme Experience. International Journal of Rural Management. 2006; 2(2): 213–226. Publisher Full Text\n\nOmonijo DO, Toluwase SOW, Oludayo OA, et al.: Impacts of Agricultural Development Programme(ADP) on Rural Dwellers in Nigeria: A Study of Isan-Ekiti. International Research Journal of Finance and Economics. 2014; 128: 41–55. Reference Source\n\nTunji Titilola ST: Environment and sustainable agricultural development in Nigeria(online). 2001. Reference Source\n\nUgwu DS: Contributions of agricultural development programmes (ADPs) to rural livelihood and food security in Nigeria. Agricultural Journal. 2007; 2(4): 503–510. Reference Source\n\nUmeh OJ, Ekumankama OO, Nwachukwu I, et al.: Comparative performance evaluation of the Agricultural development programmes of Abia and Enugu states, Nigeria. J Agric Ext. 2015; 19(2): 108–114. Publisher Full Text" }	[ { "id": "43240", "date": "15 Mar 2019", "name": "Philip Olasupo Alege", "expertise": [ "Reviewer Expertise Macroeconomics", "Dynamic Stochastic General Equilibrium Models" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 literature review is not adequate. The literature review lacks depth and spread.\nGeneral Comments: The author should provide enough review of studies in similar areas covering the theoretical explanations. Also, the review should be considered over a period of time. That is, the review should focus on old and recent studies in the subject area so as to clearly situate the gaps to be filled in the literature.\n\nFADAMA The author provide the full meaning of the acronym when it is mentioned for the first time.\n\nGrammatical Errors The authors should cross-check the document for errors. Example: author wrote \"where\" instead of \"were\"\n\nSpelling Errors of Authors Cited The authors should check the correct name of an author on page 6. The author wrote \"Cochranor\" instead of \"Cochrane\"\n\nMathematical Errors The authors should rewrite the mathematical formula correctly in Page 6, Paragraph 3\n\nAdditional Information: Hypotheses statements The statements of hypotheses in the work are not correctly stated. For example, Hypothesis One states that: Agricultural Extension Services of Edo Agricultural Development Programme (ADP) vs. crop development\nHowever, the statement is not testable. Hypothesis should be stated in the null and alternative form to be testable.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4482", "date": "18 Mar 2019", "name": "Henry INEGBEDION", "role": "Author Response", "response": "We accept responsibility for the editing (grammatical errors) and promise to review them. we may also do a minor update of the literature in line with the reviewer's comments. However, we do not think it is compulsory to state the null and alternative hypotheses to confirm statistical testability. often times, it is the null hypothesis that is tested in a study, not the null and alternative. Once the null hypothesis is formulated, the alternative hypothesis is implied, as the null hypothesis often denies the existence of the relationship between two or more variables." }, { "c_id": "4613", "date": "13 May 2019", "name": "Henry INEGBEDION", "role": "Author Response", "response": "Comment: The literature review is not adequate. The literature review lacks depth and spread.Response: Additional Empirical studies, find below Auta and Dafwang (2010) investigated the status and policy of ADPs in Nigeria. they found that over sixty three percent of the ADPs had weak or very weak funding status while over twenty two percent had good to excellent status. Furthermore, most of the ADPs had reduced their extension workers in recent times due to poor fundingChukwuemeka and Nzewi (2011) investigated World Bank sponsored Agricultural Development Project to find out the extent to which the programme had achieved set objectives. Survey and questionnaire served as research design and instrument. Findings showed that beneficiaries were excluded from design, planning and implementation of project; a development that was perceived as undesirable. Besides, political considerations, rather than expertise and professionalism was found to characterise the recruitment of extension staff; and the joint financiers (World Bank, Federal and State governments of Nigeria) were not fulfilling their financial obligations as at when due. Okuokenye and Okoedo-Okojie (2014) investigated activities of extension agents on agricultural loans and inputs supply programme participant farmers’ rice output/income. Questionnaire served as the instrument of data collection from a sample of 60 randomly selected extension farmers and 80 participant and 80 non-participant farmers’. Results showed that the extension agents made significant impact on output and income. The major constraints to the implementation of the programme were found to be restricted coverage of farms and wrong selection of participants Naswem and Ejembi (2017) historically examined the extent to which the agricultural extension programme has enhanced transition from subsistence to commercial agriculture in Nigeria. they observed that the programme had made some significant impact but was currently constrained by dwindling oil revenues and, most importantly, the absence of a coherent extension policy Comment: The literature review is more like an annotated biography. I would have liked to have seen some discussion of the limitations of the existing research. There is no discussion of how the current research adds to or improves upon the existing literature. Response:2.2.1. Research Gap The empirical review reveals that adequate empirical literature abound on agricultural financing through the extension services of ADP in Nigeria. Most of the studies agree that the ADPs have made significant impact on agricultural production in Nigeria, especially in the a areas of increased agricultural output and income as well as improved rural livelihood (Ammani, Auta & Aliyu, 2010; Olujenyo, 2006; Ugwu, 2007; Adamu & Mohammed, 2009; Omonijo, Toluwase, Oludayo & Uche, 2014; and Okuokenye & Okoedo-Okojie, 2014). However, not all the objectives of the programme have been successful. Specifically, the provision of credit facilities (Omonijo, Toluwase, Oludayo & Uche (2014) and infrastructural developments (Adamu & Mohammed, 2009). Furthermore, despite the perceived positive impact of the ADP in agricultural outputs and income, findings also indicate that there are challenges currently being faced by the programme in a significant number of the states where it is being implemented. These challenges could erode the credibility and “Goings Concern” of the implementation of the project if urgent steps are not taken to mitigate the challenges. The major challenges include inadequate funding, mainly as a result of the inability of critical stakeholders (World Bank, federal government and State governments) to fulfill their financial obligations to the programme as and when due (Ugwu, 2007; Ammani, Auta & Aliyu, 2010; Chukwuemeka & Nzewi, 2011) Omonijo, Toluwase, Oludayo & Uche, 2014; Auta & Dafwang, 2010; and Okuokenye & Okoedo-Okojie, 2014) as well as politicization of the selection of participants in the implementation of the programme by government (Ugwu, 2007; Chukwuemeka & Nzewi, 2011; and Okuokenye & Okoedo-Okojie, 2014).In view of the foregoing, the need to re-awaken the interest of stakeholders on the actualization of the objectives of the ADP becomes very important. Besides, the perceived insignificant impact on infrastructural development should attract the interest for stakeholders for adequate intervention. The constraints to the continued implementation of the scheme also raise some questions to stakeholders. This study attempts to fill these gaps CommentGrammatical Errors The authors should cross-check the document for errors. Example: author wrote \"where\" instead of \"were\" Spelling Errors of Authors Cited: The authors should check the correct name of an author on page 6. The author wrote \"Cochranor\" instead of \"Cochrane\" Response This has been corrected on page 4 in the following paragraph: The FADAMA programme of the ADP, which began in the north has also taken off in the south and is currently undertaken by Edo ADP. FADAMA involves interventionist programmes, using relevant methodologies in areas where water is close to the surface of the earth. The frequency of . .. This has been corrected in the paragraph: The value for Z is obtained from the normal distribution table, from the area under the normal curve (Cochrane, 1963; and Agbadudu, 2007). Comment: Additional Information: Hypotheses statementsThe statements of hypotheses in the work are not correctly stated.For example, Hypothesis One states that: Agricultural Extension Services of Edo Agricultural Development Programme (ADP) vs. crop developmentResponseThe hypotheses have been reformulate to reflect this correctionHypotheses Agricultural extension services of Edo ADP have not significantly affected crop development in the communities Agricultural extension services of Edo ADP have not significantly affected farm development in the communities Agricultural extension services of Edo ADP have not significantly affected the development of infrastructural facilities in the communities Agricultural extension services of Edo ADP have not significantly influenced reduction in the level of unemployment in the communities Comment: Mathematical ErrorsThe authors should rewrite the mathematical formula correctly in Page 6, Paragraph 3ResponseThe correct formula is presented below:n = ,." }, { "c_id": "4639", "date": "13 May 2019", "name": "Henry INEGBEDION", "role": "Author Response", "response": "This revised version has taken care of virtually all the errors that were identified by the reviewers. We have also revised the literature in line with the reviewer's comments and included a section on gap analysis. The hypotheses have now been stated in the null form to address the concern raised by the first reviewer. We have no competing interests to disclose." } ] }, { "id": "47572", "date": "29 Apr 2019", "name": "David L. Sjoquist", "expertise": [ "Reviewer Expertise Public economics", "policy evaluation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 literature review is more like an annotated biography. I would have liked to have seen some discussion of the limitations of the existing research. There is no discussion of how the current research adds to or improves upon the existing literature. While the survey questions are available, it would be very helpful if the questions were made part of the paper. Furthermore, a table of the responses would be helpful for the reader. The existing table only provides the mean response, but the distribution of the responses would be very helpful in understanding the results.\nA minor point, but the first three and second three tables could each be combined into one table, saving some space. The table numbers referred to in the text do not match the table numbers. Tables 5 and Table 6 both refer to educational attainment, while the text discusses location and refers to table 5. There is a reference to Table 5.1, but there is no table 5.1 in the paper. The table titles and hypotheses statements could be improved; the current title makes it seem that the analysis is pitting the ADP program against (“versus”), for example, crop development.\n\nThe survey asks multiple questions on some issues, but the authors do not explain what question or questions are used to calculate the means reported in tables. The authors misstate the what the tables show. For example, they state that “…ADP have significantly affected…” What the survey can tell us, is that these services are perceived to have affected, say farm development, which is substantially different from knowing that the services actually affected, say farm development. The authors note when the effects are statistically significant. But, it would be important to note whether the effects are economically significant.\nThe paper suggests some policy changes. However, the research reported in the paper does not provide support for (or against) the various policy recommendation. For example, the authors state that the authorities should re-strategize and reengineer the extension services. But there are no questions in the survey that suggest that such changes are necessary or would improve the services.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4614", "date": "13 May 2019", "name": "Henry INEGBEDION", "role": "Author Response", "response": "The literature review is more like an annotated biography. I would have liked to have seen some discussion of the limitations of the existing research. There is no discussion of how the current research adds to or improves upon the existingResponse: Additional Empirical studies, find below Auta and Dafwang (2010) investigated the status and policy of ADPs in Nigeria. they found that over sixty three percent of the ADPs had weak or very weak funding status while over twenty two percent had good to excellent status. Furthermore, most of the ADPs had reduced their extension workers in recent times due to poor fundingChukwuemeka and Nzewi (2011) investigated World Bank sponsored Agricultural Development Project to find out the extent to which the programme had achieved set objectives. Survey and questionnaire served as research design and instrument. Findings showed that beneficiaries were excluded from design, planning and implementation of project; a development that was perceived as undesirable. Besides, political considerations, rather than expertise and professionalism was found to characterise the recruitment of extension staff; and the joint financiers (World Bank, Federal and State governments of Nigeria) were not fulfilling their financial obligations as at when due. Okuokenye and Okoedo-Okojie (2014) investigated activities of extension agents on agricultural loans and inputs supply programme participant farmers’ rice output/income. Questionnaire served as the instrument of data collection from a sample of 60 randomly selected extension farmers and 80 participant and 80 non-participant farmers’. Results showed that the extension agents made significant impact on output and income. The major constraints to the implementation of the programme were found to be restricted coverage of farms and wrong selection of participants Naswem and Ejembi (2017) historically examined the extent to which the agricultural extension programme has enhanced transition from subsistence to commercial agriculture in Nigeria. they observed that the programme had made some significant impact but was currently constrained by dwindling oil revenues and, most importantly, the absence of a coherent extension policy Comment: The literature review is more like an annotated biography. I would have liked to have seen some discussion of the limitations of the existing research. There is no discussion of how the current research adds to or improves upon the existing literature. Response:2.2.1. Research Gap The empirical review reveals that adequate empirical literature abound on agricultural financing through the extension services of ADP in Nigeria. Most of the studies agree that the ADPs have made significant impact on agricultural production in Nigeria, especially in the a areas of increased agricultural output and income as well as improved rural livelihood (Ammani, Auta & Aliyu, 2010; Olujenyo, 2006; Ugwu, 2007; Adamu & Mohammed, 2009; Omonijo, Toluwase, Oludayo & Uche, 2014; and Okuokenye & Okoedo-Okojie, 2014). However, not all the objectives of the programme have been successful. Specifically, the provision of credit facilities (Omonijo, Toluwase, Oludayo & Uche (2014) and infrastructural developments (Adamu & Mohammed, 2009). Furthermore, despite the perceived positive impact of the ADP in agricultural outputs and income, findings also indicate that there are challenges currently being faced by the programme in a significant number of the states where it is being implemented. These challenges could erode the credibility and “Goings Concern” of the implementation of the project if urgent steps are not taken to mitigate the challenges. The major challenges include inadequate funding, mainly as a result of the inability of critical stakeholders (World Bank, federal government and State governments) to fulfill their financial obligations to the programme as and when due (Ugwu, 2007; Ammani, Auta & Aliyu, 2010; Chukwuemeka & Nzewi, 2011) Omonijo, Toluwase, Oludayo & Uche, 2014; Auta & Dafwang, 2010; and Okuokenye & Okoedo-Okojie, 2014) as well as politicization of the selection of participants in the implementation of the programme by government (Ugwu, 2007; Chukwuemeka & Nzewi, 2011; and Okuokenye & Okoedo-Okojie, 2014).In view of the foregoing, the need to re-awaken the interest of stakeholders on the actualization of the objectives of the ADP becomes very important. Besides, the perceived insignificant impact on infrastructural development should attract the interest for stakeholders for adequate intervention. The constraints to the continued implementation of the scheme also raise some questions to stakeholders. This study attempts to fill these gapsCommentWhile the survey questions are available, it would be very helpful if the questions were made part of the paper. Furthermore, a table of the responses would be helpful for the reader.ResponseTable A Demographic Distribution of respondents Age Category F %Under 30 Years 12 4.831-40 Years 75 30.241-50 Years 105 42.350 Years and above 56 22.6Total 248 100 Sex Male 169 68.1 Female 31.9 31.9 Total 248 100 Marital Status Married 191 77 Single 38 15.3 Widowed 15 6 Divorced 4 1.6 Total 248 100 Highest Education SSCE 113 45.6 ND/NCE 82 33.1 HND/First Degree 51 20.6 Higher Degree 2 8 Total 248 100 Local Govt. Area Oredo 82 33.1 Egor 90 36.3 Ikpoba Okha 76 30.6 Total 248 100 Table B Response to Items on Research Problem Variable Item SD D NV A SA Farm and Crop Development Q1 10 70 58 62 28 Q2 14 75 54 79 26 Q3 9 72 61 81 25 Infrastructural Development Q4 20 76 53 75 24 Q5 14 73 57 79 24 Q6 21 71 57 70 29 Reduction in Unemployment Q7 17 75 55 78 23 Q8 18 24 57 78 21 Comment Are the conclusions drawn adequately supported by the results? NoResponseAn additional conclusion not initially captured is presented below:The pattern of intervention in some of the areas was significantly different fom others, thus the impact of the extension programmes of ADP are not uniform across locations but vary from location to location" } ] } ]	1	https://f1000research.com/articles/7-1833
https://f1000research.com/articles/8-754/v1	29 May 19	{ "type": "Research Article", "title": "Sleep monitoring with the Apple Watch: comparison to a clinically validated actigraph", "authors": [ "Sirinthip Roomkham", "Michael Hittle", "Joseph Cheung", "David Lovell", "Emmanuel Mignot", "Dimitri Perrin", "Sirinthip Roomkham", "Michael Hittle", "Joseph Cheung", "David Lovell", "Emmanuel Mignot" ], "abstract": "Background: We investigate the feasibility of using an Apple Watch for sleep monitoring by comparing its performance to the clinically validated Philips Actiwatch Spectrum Pro (the gold standard in this study), under free-living conditions. Methods: We recorded 27 nights of sleep from 14 healthy adults (9 male, 5 female). We extracted activity counts from the Actiwatch and classified 15-second epochs into sleep/wake using the Actiware Software. We extracted triaxial acceleration data (at 50 Hz) from the Apple Watch, calculated Euclidean norm minus one (ENMO) for the same epochs, and classified them using a similar algorithm. We used a range of analyses, including Bland-Altman plots and linear correlation, to visualize and assess the agreement between Actiwatch and Apple Watch. Results: The Apple Watch had high overall accuracy (97%) and sensitivity (99%) in detecting actigraphy-defined sleep, and adequate specificity (79%) in detecting actigraphy defined wakefulness. Over the 27 nights, total sleep time was strongly linearly correlated between the two devices (r=0.85). On average, the Apple Watch over-estimated total sleep time by 6.31 minutes and under-estimated Wake After Sleep Onset by 5.74 minutes. The performance of the Apple Watch compares favorably to the clinically validated Actiwatch in a normal environment. Conclusions: This study suggests that the Apple Watch could be an acceptable alternative to the Philips Actiwatch for sleep monitoring, paving the way for larger-scale sleep studies using Apple’s consumer-grade mobile device and publicly available sleep classification algorithms. Further study is needed to assess longer-term performance in natural conditions, and against polysomnography in clinical settings.", "keywords": [ "Apple Watch", "Sleep Study", "Actigraphy" ], "content": "Introduction\n\nGood sleep is vital for our health and wellbeing. Without it, our body and mind function poorly, with consequences that include risk of obesity1, diabetes2 and cardiovascular disease3. Polysomnography (PSG) is currently the gold standard method to monitor sleep. During PSG, subjects spend a night in a dedicated sleep lab, hooked up to a range of devices to measure physiological signals. However, information-rich PSG is expensive, laborious and intrusive. Because subjects are attached to many electrodes, sleep is disturbed by the recording, so that it is better at looking at qualitative abnormalities such as sleep apnea or narcolepsy.\n\nWidespread adoption of smartphones, smartwatches and fitness devices opens up new opportunities for monitoring sleep and physical activity. Many of these consumer devices come with a built-in accelerometer, gyroscope, magnetometer, and in some cases, a heart rate sensor—more sensors than wrist-worn actigraphs, which only use an accelerometer to measure movement to infer sleep and wake states. Nevertheless, actigraphs are regarded as useful tools in clinical practice when measuring sleep and wakefulness in normal living environments, especially in the context of assessing regularity and overall diurnal fluctuations4–7.\n\nThe affordability of consumer-grade mobile devices has driven their popularity and the abundance of applications (“apps”) available—including apps for health and sleep. These devices have the potential to be used in sleep studies and to inform clinical diagnosis. However, their performance needs to be properly validated in comparison to accepted methodologies such as actigraphy and PSG. This is difficult because the sleep classification methods (e.g., algorithms and analysis techniques) used in consumer-grade devices are proprietary, so the relationship between the underlying physiological measurements and the sleep state reported by an app is unclear.\n\nWe chose to investigate the feasibility of the Apple Watch as a sleep monitoring device because the manufacturer allows software developers to access the device’s triaxial accelerometer data. To the best of our knowledge, this is the first study to compare the Apple Watch against an actigraph.\n\n\nMethods\n\nFigure 1 sets out the framework for validating the Apple Watch against an actigraph. First, raw acceleration measurements are collected from each device and transformed into activity counts for the actigraph, and the “Euclidean Norm Minus One” (ENMO) for the Apple Watch. We then explore statistical relationships between activity counts and ENMO. Next, we determine a threshold to classify each epoch of ENMO values as “sleep” or “wake” and evaluate the sleep outputs of the two devices using Pearson correlation and Bland-Altman plots.\n\nIn total, 14 healthy participants (9 males, 5 females) were recruited by word of mouth and direct approach. Data were recorded from April to May 2018, participants wore the two devices for two consecutive nights at home. The inclusion criterion for participants was age of at least 18 years old. Exclusion criteria were a previously diagnosed sleep disorder, or any condition that would lead to difficulty/discomfort while wearing the devices. The Queensland University of Technology Human Research Ethics Committee (#1800000242) approved all procedures, and all participants gave their signed consent prior participating the study. They were asked to wear both the Apple Watch and Actiwatch on their non-dominant wrist for two consecutive nights and sleep as they normally would. All wearable devices were then returned for data extraction. One participant forgot to charge the Apple Watch, so we lost one night of data and were left with 27 nights from 14 participants.\n\nThis study was designed as a proof of concept for whether it is possible to use the Apple Watch for sleep monitoring. Power calculations are appropriate when the distribution of the underlying data is known (and ideally, normal). At that point in time, this was not applicable given that there was no prior study for us to confidently characterize the distribution of the differences between the measurements obtained using different platforms. Sample size was therefore determined pragmatically8.\n\nTable 1 shows specifications of the two wrist-worn devices used in this study: the Apple Watch Series 1 (Apple Inc., California, United States) and the Actiwatch Spectrum Pro (Philips, Bend OR). The Apple Watch has limited data storage, so data was downloaded to an iPhone via Bluetooth. We used the Core Motion Framework to develop an app to record triaxial accelerometer data at 50 Hz. The Actiwatch Spectrum Pro is a clinical-grade actigraph used for sleep and activity monitoring. It samples accelerometer data at 32 Hz and we set a 15-second epoch for processing this raw data. Processed data were downloaded using Philips’ Actiware Software (version 6.0.9). Outputs were activity counts and sleep/wake stage at each epoch.\n\nAccelerometer. Raw acceleration data from the Apple Watch was downloaded and processed using R statistical software. We calculated ENMO, the Euclidean Norm (magnitude) of the triaxial acceleration vector A = (Ax, Ay, Az) minus 1 gravitational unit. ENMO is used widely in physical activity and sleep monitoring9–13 and defined as:\n\n\n\nWe compared the mean ENMO of each 15-second epoch against activity counts from the actiwatch. We ensured that the clocks of both devices were synchronized and compared recordings of vigorous movement applied simultaneously to both devices to check that each devices’ timestamps were in sync.\n\nSleep algorithm. Philips’ Actiware software computes the total activity counts at epoch e using a weighted sum:\n\nTotal_counts(e) = 0.04∑i=−8i=−5ae+i+0.2∑i=−4i=−1ae+i+4ae+0.04∑i=1i=4ae+i+0.2∑i=5i=8ae+i\n\nwhere ae is the activity count of epoch e. We used the shortest (15-second) epoch so that data could be converted to longer epochs if required. Each epoch was classified as sleep if its total activity counts were less than or equal to a threshold; epochs with counts above the threshold were classified as wake. Our study used a low and medium threshold (20 and 40, respectively) derived from Actiware 6.0.9 software.\n\nStatistical analysis. The R program (version 3.3.2) is used for statistical analysis and visualization. To measure agreement between Apple Watch and Actiwatch, we used a range of statistical methods including:\n\ni Calculating the Pearson Correlation between total activity counts and ENMO.\n\nii Using a receiver operating characteristic (ROC) analysis, taking the Actiwatch as ground truth.\n\niii Using Bland-Altman plots to measure the agreement between two measurements by quantifying the mean bias and constructing an agreement interval.\n\niv Computing the overall accuracy and performance the devices’ sleep/wake classifications using a confusion matrix, again taking the Actiwatch as ground truth.\n\nThe confusion matrix of sleep-wake classifications has four outcomes: true positive (TP), true negative (TN), false positive (FP), and false negative (FN), with sleep positive, and wake negative. Using these outcomes, we calculated accuracy, sensitivity, specificity, and F1 score as defined in Table 214.\n\nWe also calculated measures of sleep quality of interest in sleep studies: total sleep time (TST), wake after sleep onset (WASO), and number of awakenings. TST is the total duration of epochs classified as sleep; WASO is the total duration of wake epochs. Number of awakenings is the number of wake events of at least 30 seconds duration15.\n\n\nResults\n\nFigure 2 displays measurements over the first night of randomly selected participant: overall patterns of Actiwatch and Apple Watch are very similar with clearly aligned periods of movement. For example, around 22:10, both activity counts and ENMO were quite active with high peaks, then both signals gradually declined. During a sleep period from 02:00–02:30, both features remained steady with no obvious movements. Raw measurements are available as Underlying data16.\n\nENMO is shown in turquoise (left) and Activity Counts in pink (right).\n\nPearson correlation was computed to assess the relationship between activity counts and ENMO shown in Figure 3. Overall, there was strong, positive correlation between activity counts and ENMO (r = 0.85, nights = 27, p<0.001). There were similar patterns across 15-second, 30-second, and 60-second epochs. We used 15-second-epochs in the remainder of this analysis.\n\nThe diagonal line is the standardized major axis fit17 of activity counts and mean ENMO, constrained to pass through the origin.\n\nUnlike the Actiwatch, there is no pre-established wake threshold for the Apple Watch ENMO data. To identify an optimal threshold, we isolated 11 nights without any missing data, and created all the possible training sets containing k nights, where k varies from 1 to 10. For instance, for k = 1 or k = 10 there are 11 such training sets, while for k = 5 or k = 6 there are 462 sets. In total, we considered 2046 distinct training sets. For each of these sets, we identified the threshold that maximized the F1 score on the nights not used for training. Figure 4 shows how this threshold converges to 0.0608 when comparing against the medium Actiwatch threshold. This ‘optimal’ threshold was used for the rest of our analysis. A similar pattern was observed for the low Actiwatch threshold, with the Apple Watch threshold converging to 0.0523. We also measured the impact of the threshold choice through a ROC curve for the medium Actiwatch setting (Figure 5).\n\nAs the number of training nights increase, the distributions converge around a value of 0.0608.\n\nOverall, there is good agreement between the Apple Watch and Actiwatch for both medium and low setting thresholds. Significantly, the ability to detect sleep (sensitivity) are higher than 98%. However, the ability to detect awake ranges from 60% to 79% for both thresholds. The overall accuracy and F1 score were consistent for both settings. The results are summarized in Table 3.\n\nFigure 6 plots the difference versus the mean of TST, WASO and numbers of awakenings for Apple Watch and Actiwatch. We used a 15-second epoch and medium threshold for our main analysis. For TST, most nights were within the limits of agreement and close to perfect agreement (the black line, Figure 6a)—three nights of TST were in perfect agreement. Only one night was outside the agreement intervals and was overestimated TST by 30 minutes. The overall bias of TST was 6.31 minutes.\n\nThe red dashed lines represent in the upper and lower agreement limits (95% confidence intervals). (a) Total sleep time (minutes). (b) Wake after sleep onset (minutes). (c) Number of awakenings.\n\nIn terms of WASO, differences fall mostly within the levels of agreements (Figure 6b)—two nights were in perfect agreement. One night stood outside the agreements which an underestimation of WASO by 30 minutes. This night was the same night that we found in TST. The overall bias of estimating WASO was -5.74 minutes.\n\nFor the total number of awakenings, the overall bias was -4.56. Only one night was in perfect agreement but all nights lie within the upper and lower agreement levels (Figure 6c).\n\n\nDiscussion\n\nThis study compares measurements of sleep and wake obtained from Apple Watch, a consumer-grade device, and Philips Actiwatch Spectrum Pro, a clinically validated actigraphy device, in a set of healthy adults. We found that ENMO and activity counts were highly correlated. By using a combination of ten-night training data and ROC plot, we identified an optimal threshold for Apple Watch ENMO data in comparison to the Actiwatch for both medium and low settings (Table 3).\n\nIn the medium threshold setting, the sleep parameters of TST, WASO, and number of awakenings were comparable to that of the Actiwatch with no significant differences. The discrepancy between the two measurements appeared to be clinically acceptable as a difference of TST and WASO did not exceed 30 minutes6,18. The Apple Watch performs best in comparison to the Actiwatch at medium threshold, consistent with Quante’s recommendation19.\n\nTo the best of our knowledge, this is the first study to evaluate the Apple Watch, a popular-consumer grade device, against a clinical-grade actigraph device for sleep monitoring. We have compared the two devices at high resolution (i.e., 15-second epochs) and low-resolution sleep parameters (i.e., TST, WASO, and number of awakenings). A similar study compared a consumer fitness tracking device (Fitbit charge HR) against Philips’ Actiwatch 2: the accuracy of sleep parameters was good20. However, Montgomery-Downs et al. suggested that both Fitbit and Actiwatch tended to have limited specificity21. Therefore, care must be paid in validation. Furthermore, both studies compared only low-resolution sleep parameters due to the limited information of the type of feature, and proprietary sleep algorithm22. Our study provides additional support for high-resolution data (i.e., ENMO), which could be further used in a sleep-wake algorithm validated against gold standard PSG.\n\nWe note that ENMO and activity counts are dominated by very small values (i.e., points near or at (0, 0)), as shown in Figure 3. This is a consequence of doing a study in which participants are moving very little for relatively long periods. While this is reasonable in assessing whether the Apple Watch produces comparable results to the Actiwatch for sleep monitoring, this study cannot draw conclusions about whether the Apple Watch is comparable to the Actiwatch across a broader range of activity levels (e.g., during exercise). The main benefit of assessing sleep using a well-known consumer-grade device lies in increasing the opportunity for longitudinal studies in a wider population. Over 30 million Apple Watches have been sold23. The availability of advanced sensor technology in smart watches (e.g., heart-rate sensors) opens up possibilities for improved sleep monitoring with consumer wearable devices.\n\nWe faced some practical challenges in implementing sleep monitoring on the Apple Watch, including constraints of power, memory management, data transfer, and sensor capabilities. In total, 16 nights had missing data from the Apple Watch. In each of these nights, data was missing in one contiguous block of 20–60 minutes in duration. We needed to impute data based on the average of previous and next available data. At this stage, the cause of the missing data is still to be determined.\n\nRecording was limited to two consecutive nights per participant due to memory and data transfer constraints. With these limitations in mind, we suggest future studies are needed to carefully monitor and deal with data loss where more nights of recording are investigated. Lastly, our study assessed only one sleep-wake algorithm based on the Cole-Kripke algorithm17; future studies could assess other sleep-wake algorithms that use a combination of weighted sum activity with the cut-off wake threshold to classify sleep or wake stages15.\n\n\nConclusion\n\nOur study lays down a foundation for using accelerometer data of consumer-grade devices for sleep monitoring. Our experiments show that Apple Watch provides sleep measures comparable to the Philips Actiwatch, a clinical gold standard, with greatest similarity at the medium threshold of activity counts. These findings increase our confidence in using the consumer grade Apple Watch for sleep monitoring and open up possibilities for much larger-scale sleep studies. We also hope this work will serve as a basis for sleep clinicians in the use of data extracted from this device in their patients.\n\nTo further our research, we now intend to compare the Apple Watch against PSG and consider incorporating other types of physiological sensors from consumer-grade devices (e.g. heart-rate sensor). These findings add to a growing body of literature on the use of consumer-based accelerometer for sleep studies and will assist other researchers in establishing validation parameters for the use of other types of consumer devices.\n\n\nData availability\n\nQUT Research Data Finder: Sleep Data. https://doi.org/10.25912/5cc28f62e81ad16.\n\nUnderlying data are contained within SleepDataset.zip. There are 27 csv files in this archive, with each file corresponding to a single night of data. The files have four columns: timestamp, Actiwatch activity counts, Actiware classification (1 for wake, 0 for sleep), and ENMO value calculated from the Apple Watch data. Each row corresponds to the data for a 15-second epoch. Dates have been modified to preserve privacy, with times are unchanged.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nDevices were funded by internal funds from the Queensland University of Technology (equipment grant from the Science and Engineering Faculty for the Philips Actiwatch Spectrum Pro and Actiware Software, ECARD grant for the Apple Watch).\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\nWatanabe M, Kikuchi H, Tanaka K, et al.: Association of short sleep duration with weight gain and obesity at 1-year follow-up: a large-scale prospective study. Sleep. 2010; 33(2): 161–167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpiegel K, Knutson K, Leproult R, et al.: Sleep loss: a novel risk factor for insulin resistance and Type 2 diabetes. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Hees VT, Fang Z, Langford J, et al.: Autocalibration of accelerometer data for free-living physical activity assessment using local gravity and temperature: an evaluation on four continents. J Appl Physiol (1985). 2014; 117(7): 738–744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRowlands AV, Yates T, Davies M, et al.: Raw Accelerometer Data Analysis with GGIR R-package: Does Accelerometer Brand Matter? Med Sci Sports Exerc. 2016; 48(10): 1935–1941. PubMed Abstract \| Publisher Full Text\n\nCheung J, Zeitzer JM, Lu H, et al.: Validation of minute-to-minute scoring for sleep and wake periods in a consumer wearable device compared to an actigraphy device. Sleep Science Practice. 2018; 2(1): 11. Publisher Full Text\n\nSaito T, Rehmsmeier M: The precision-recall plot is more informative than the ROC plot when evaluating binary classifiers on imbalanced datasets. PLoS One. 2015; 10(3): e0118432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalland BC, Kennedy GJ, Mitchell EA, et al.: Algorithms for using an activity-based accelerometer for identification of infant sleep-wake states during nap studies. Sleep Med. 2012; 13(6): 743–751. PubMed Abstract \| Publisher Full Text\n\nRoomkham S, Michael H, Joseph C, et al.: Sleep study comparison using the Apple Watch and the Philips Actiwatch. Queensland University of Technology. 2019.\n\nSadeh A, Lavie P, Scher A, et al.: Actigraphic home-monitoring sleep-disturbed and control infants and young children: a new method for pediatric assessment of sleep-wake patterns. Pediatrics. 1991; 87(4): 494–499. PubMed Abstract\n\nWerner H, Molinari L, Guyer C, et al.: Agreement rates between actigraphy, diary, and questionnaire for children's sleep patterns. Arch Pediatr Adolesc Med. 2008; 162(4): 350–358. PubMed Abstract \| Publisher Full Text\n\nQuante M, Kaplan ER, Cailler M, et al.: Actigraphy-based sleep estimation in adolescents and adults: a comparison with polysomnography using two scoring algorithms. Nat Sci Sleep. 2018; 10: 13–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee HA, Lee HJ, Moon JH, et al.: Comparison of Wearable Activity Tracker with Actigraphy for Sleep Evaluation and Circadian Rest-Activity Rhythm Measurement in Healthy Young Adults. Psychiatry Investig. 2017; 14(2): 179–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMontgomery-Downs HE, Insana SP, Bond JA: Movement toward a novel activity monitoring device. Sleep Breath. 2012; 16(3): 913–917. PubMed Abstract \| Publisher Full Text\n\nRoomkham S, Lovell D, Cheung J, et al.: Promises and Challenges in the Use of Consumer-Grade Devices for Sleep Monitoring. IEEE Rev Biomed Eng. 2018; 11: 53–67. PubMed Abstract \| Publisher Full Text\n\nIdc: IDC Forecasts Shipments of Wearable Devices to Nearly Double by 2021 as Smart Watches and New Product Categories Gain Traction. 2017. Reference Source" }	[ { "id": "50110", "date": "10 Jul 2019", "name": "Sean P. A. Drummond", "expertise": [ "Reviewer Expertise Sleep", "insomnia", "validation of wearables", "sleep deprivation", "cognitive performance", "mental health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this this paper, the authors attempt to optimize an algorithm to convert accelerometer data from the Apple Watch to match that provided by a research grade actigraph, the Actiwatch Spectrum Pro. This is a proof of concept study, and thus has a fairly small sample size. Overall, the analyses show the authors are able to convert the Apple Watch data into the same epoch-by-epoch sleep/wake data as the Atiwatch with impressive sensitivity and specificity. Not surprisingly, then, the Total Sleep Time and Wake After Sleep Onset values from the Apple Watch also match the Actiwatch quite well. While I believe this algorithm development is a valuable addition to the literature, there are several areas where the manuscript could be improved. In particular, I believe the authors oversell their results and over state conclusions. There are also some areas where the reader would benefit from more detail or clarity. Overall, I am extremely supportive of this line of work and this paper. However, given the scepticism of this line of work among many in the field, it is critical research on consumer sleep trackers is conducted with strong methodology, is reported clearly, and is interpreted in line with the findings. My specific comments, below, are written with that in mind:\n\nMy biggest concern about the paper is the authors focus on the claim the Apple Watch can score sleep/wake the same as the Actiwatch. However, fundamentally, this is not what they have shown. They have shown their algorithm can faithfully replicate the sleep/wake designation made by the Actiwatch on a 15-sec basis. While this would be expected to, and indeed does, produce largely similar all-night sleep measures, that is not the same thing as validating the all-night sleep measures produced by Apple Watch. That can only be done with a PSG validation study. I appreciate the author never explicitly claim this is a “validation” study. Nonetheless, the language they use throughout certainly implies such. I would encourage the authors to refocus the paper around the finding related to their algorithm being optimized to produce the same epoch-level decision as the Actiwatch makes. They can still show the TST and WASO data as evidence this likely translates into all-night sleep measures, but the latter should not be the take home message. This concern propagates through several of the following comments, as well.\n\nAbstract: I would encourage the authors to focus the Results section on the algorithm success, not on TST and WASO. They should also remove the claim the Apple Watch “…the Apple Watch could be an acceptable alternative to the Philips Actiwatch for sleep monitoring…”, as this cannot be shown without a formal PSG validation study.\n\nIntroduction: While everyone agrees PSG has serious limitations, I would not categorize sleep apnea and narcolepsy as \"qualitative\" abnormalities. They are indeed quantified in a very specific way. It would be more fair to simply say PSG is better suited for use on a single night to detect sleep disorders.\n\nIntroduction: The authors should clearly state the Aims of the study at the end of the Intro. As stated above, I believe the primary Aim should be to determine if ENMO can provide the same epoch-level sleep/wake decision as that produced by Actiwatch. The TST and WASO assessments should be secondary or exploratory Aims. This would both reflect the supporting role they play to the main Aim, as well as the fact the study is underpowered to detect differences between the two devices in all-night sleep measures.\n\nMethods: The reader would benefit from a better description of the sample. What was the age mean, standard deviation, and range? What was the sex ratio? How did the authors screen for sleep disorders? Were there any medical/psychiatric diagnoses? In my view, it may not matter what the exact answers are, as the main purpose here is to see if the Apple Watch can detect motion to the same extent as the Actiwatch. In that regard, it should not really matter who is producing that motion. One the other hand, most actigraphs and consumer wearables perform differently in different populations. Thus, generalizability may well be affected by the demographics of the sample studied here.\n\nMethods: Participants wore both devices simultaneously. Did the authors counter-balance the order on the wrist across participants to control for possible effects on motion?\n\nMethods: It would provide additional clarity if the authors would link each analysis to an Aim. It was not until I had finished the Results that I realised why analysis 1 and 2 were necessary. This confusion also reflects the fact the paper is written as if validating the TST and WASO measures are the main Aim (for which the first two analyses are not necessary), when in fact it is not.\n\nMethods: I am not able to comment thoroughly on the mathematical procedures used to optimize Apple Watch activity counts so they produce the same sleep/wake decision as Actiwatch. I would suggest the authors ask a modeller or other quantitative specialist to review that information.\n\nMethods: The authors report in the Discussion that they impute missing date from Apple Watch. This information should be in the Methods. Moreover, they should very clearly state how many nights and how many unique participants had missing date, as well as the mean/standard deviation and range of the missing data length. Across papers examining consumer wearables, it is becoming increasingly clear they all have problems with not recording data for chunks of time. It is critical for users of these devices to be made aware of this. Finally, I would encourage the authors to redo their analyses dropping these missing data, rather than imputing them. A lot can happen in 20-60 minutes of the night, especially given some of the nights appeared to only have ~5 hours of sleep (per Bland Altman plot). It is a faulty assumption to think the epoch before and after an hour long blank period (or even a 20 minute blank period) can substitute for every epoch within that blank period.\n\nResults: Did the authors examine night-to-night variability or test-retest reliability of the Apple Watch, given they had 2 nights for most people? If a consumer device is to be used for long periods of time in the field (by a consumer, researcher, or clinician), it is important to know if the device reliably produces the same accuracy from night to night.\n\nResults: The Bland-Altman plots seem to suggest the Apple Watch shows proportional bias, at least for TST and WASO (i.e, the device’s detection of motion, and thus sleep, becomes less accurate with lower TST and greater WASO). The authors should report proportional bias analyses for each plot, and discuss implications of the findings.\n\nDiscussion: The authors compare their findings to a single paper using a different actigraph. It is not clear why this specific paper was chosen as particularly relevant. Could they authors please clarify that? While the literature on consumer wearables is not particularly large, one would have expected the authors to put their findings into a broader context. For example, the authors could reference a recent review by de Zambotti1 to compare their findings more broadly.\n\nIt seems to me the biggest limitation of this study is the limited storage capacity and battery life of the Apple Watch. If I understand correctly, the device was only worn during the night, because it had to be charged during the day or data would be lost. Even if I misunderstand that point, the fact ”recording was limited to two consecutive nights per participant due to memory and data transfer constraints” has serious implications for the practical translation of this work: 1) This does not reflect how the typical consumer will use the Apple Watch; 2) It seem unlikely any large scale (or even small scale) study will adopt the Apple Watch (a stated goal of the authors), as it is impractical to expect participants in large cohort studies to only wear the device at night. This is especially true when there are other devices with much longer recording periods where one can also access the raw data (e.g., GeneActiv and even Fitbit with specialized software) While these points do not undermine the value of the work done here, the authors need to be more clear about these limitations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "52113", "date": "19 Aug 2019", "name": "Lillian Skeiky", "expertise": [ "Reviewer Expertise Sleep", "sleep deprivation", "wearables" ], "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\nOver the last 10 years, a large number of commercially available wearable activity trackers have been offered to the public due to the growing request for individualized health monitoring. While these commercially available devices certainly have a number of advantages compared to traditional wrist actigraphy, such as accessibility and affordability, their general validity remains unclear. Roomkham and colleagues investigated the feasibility of sleep monitoring using the commercially available Apple Watch against the Philips Actiware Spectrum pro, a research grade wrist accelerometer arguing that the performance of the Apple Watch compared favorably to the Actiwatch. This line of research is promising and should absolutely be pursued. That said, we have serious reservations regarding the methodology and analyses conducted by Roomkham and colleagues which unfortunately limits comparison between devices.\n\nOur specific comments are outlined below:\nAbstract:\nThe authors should remove the line reading, “The performance of the Apple Watch compares favorably to the clinically validated Actiwatch in a normal environment”. This is not an accurate statement as it implies that the Apple Watch has been clinically validated.\n\nThe authors should remove the line reading, “This study suggests that the Apple Watch could be an acceptable alternative to the Philips Actiwatch for sleep monitoring…” for the same reason stated in the above point.\n\nIntroduction:\nIn the first paragraph of the introduction, polysomnography (PSG) is described as qualitative for examination of OSA and narcolepsy. While we agree that PSG has certain limitations, this statement should be removed as sleep disorders are quantifiably determined using American Academy of Sleep Medicine guidelines.\n\nThe authors state that the overall aim of this study is to compare the Apple Watch against an actigraph. In actuality, the authors are comparing a variable (ENMO) based off of raw acceleration measurements produced by the Apple Watch, which should be clarified by the authors. In addition, the authors should explicitly state that they are comparing a commercial device against a validated research grade device.\n\nMethods:\nEvaluation framework:\nFurther describe Euclidean Norm Minus One (ENMO) and how this measure is comparable to Philips’ sleep algorithm.\n\nIt does not appear that sleep diaries were used in this study. Sleep diaries are integral in this line of work and are necessary for determining a number of sleep/wake variables in field-based research. Without any other supplementary information, the only reliable variable that can be used is Time in Bed. Was any supplemental information (e.g., sleep diaries, call-in times) collected from study participants? If so, this information should be presented. If not, this should be included as a limitation.\nParticipants:\nAdditional information is needed about research participants. Participants are described as “healthy”, but no further details are provided; indicate how this was ascertained. Provide additional information on how subjects were screened in general (questionnaires, physical exam, etc.)?\n\nThe authors should justify the short recording period. Traditionally, devices are worn for days to weeks at a time. For reference, a recently published abstract also using the Apple Watch had participants wear watches for > 1 month1.\nWrist-worn devices:\nDid subjects have access to the sleep statistics from the Apple Watch? The Actiwatch does not provide any feedback. It is possible that the Apple Watch could have been an unintentional intervention if they received feedback. Please address.\n\nPlease explain the Core Motion Framework in greater detail and describe the app used to record accelerometer data.\n\nPlease describe how the Actiwatch data were processed and describe data cleaning methods, if any.\n\nHow were the devices worn on the wrist? Meaning, was device placement consistent for all participants? If not, this need to be addressed as a limitation.\nData processing and analysis:\nSee the point above regarding ENMO.\n\nFurther explain why 15-second epochs were used as 30-second epochs are standard in sleep scoring.\nStatistical analysis:\nPlease describe TST and WASO as “actigraphically determined TST and WASO”. TST and WASO can only be accurately determined using PSG. In regards to field studies, a sleep diary is needed to calculate these variables.\n\nSleep Onset Latency provides useful information and should also be reported.\n\nThe citation used as the basis to quantify the number of wake events is from a study done in infants (Galland et al., 20122). Please use a reference(s) in a more relevant study in healthy adults.\n\nResults:\nThe authors imputed missing data from the Apple Watch using epochs prior to and after the missing period. Twenty to sixty minutes of a sleep period is a considerable amount of time during which significant changes may have occurred not reflected in the data used as a replacement. The authors should remove the imputed data and reanalyze using a technique designed to account for missing data.\n\nDiscussion:\nThe authors acknowledge that a major limitation of this study is the limited battery life and memory capacity of the Apple Watch. Due to these limitations, subjects wore the devices for just two consecutive nights, however this is not consistent with information from the manufacturer. Please clarify.\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? No", "responses": [] }, { "id": "49255", "date": "27 Aug 2019", "name": "Vincenzo Natale", "expertise": [ "Reviewer Expertise Sleep and individual differences in circadian rhtyhms" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of this work was to compare the measurements of sleep and wake obtained from Apple Watch and Philips Actiwatch Spectrum. To this aim, 14 participants (9 males) were asked to wear on non-dominant wrist both equipments for two consecutive nights. On the whole results show that Apple Watch performed well.\n\nThe manuscript is potentially interesting. However, there are several limitations and features that Authors should better explicit. The sample size is relatively small, but above all the number of nights is too small. Several researches show that actigraphy output reaches stability after at least seven nights.\nI feel that it is still possible to improve the manuscript. For example, Authors did not show the mean age of the sample. Authors did not explain how they calculated sleep onset. Authors did not clearly explain why they adopted a sampling rate of 15 seconds when usually, to asses sleep by actigraph, is adopted a 1 minute epoch.\n\nIn Figure 1 there is a mistake. It is reported 25 Hz but should be 32 Hz.\n\nPearson correlations are not useful. Do Authors really think that when a wrist is moving one of two devices could record no movements?\n\nThe interpretations of Bland Altman plot are a little bit questionable. Six minutes for total sleep time are few, I agree, representing more or less a variation of around 2%. However, 30 minutes for wake after sleep onset are not few because they correspond more or less to a variation of 75%. In other words, Authors have developed an algorithm very good to assess sleep (sensitivity) but less performing to evaluate wake (specificity). About this, data reported in Table 3 are very unusual because changing threshold leads to an increase in both sensitivity and specificity. Usually when one increases, the other one decreases.\n\nA last note is about the Cole-Kripke quotation at page 9. In my memory, such algorithm was implemented for another brand of actigraph (Ambulatory Monitoring). Are Authors sure about this?\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": [] } ]	1	https://f1000research.com/articles/8-754
https://f1000research.com/articles/8-752/v1	29 May 19	{ "type": "Software Tool Article", "title": "BiocPkgTools: Toolkit for mining the Bioconductor package ecosystem", "authors": [ "Shian Su", "Vincent J. Carey", "Lori Shepherd", "Matthew E. Ritchie", "Martin T. Morgan", "Sean Davis", "Shian Su", "Vincent J. Carey", "Lori Shepherd", "Matthew E. Ritchie", "Martin T. Morgan" ], "abstract": "Motivation: The Bioconductor project, a large collection of open source software for the comprehension of large-scale biological data, continues to grow with new packages added each week, motivating the development of software tools focused on exposing package metadata to developers and users. The resulting BiocPkgTools package facilitates access to extensive metadata in computable form covering the Bioconductor package ecosystem, facilitating downstream applications such as custom reporting, data and text mining of Bioconductor package text descriptions, graph analytics over package dependencies, and custom search approaches. Results: The BiocPkgTools package has been incorporated into the Bioconductor project, installs using standard procedures, and runs on any system supporting R. It provides functions to load detailed package metadata, longitudinal package download statistics, package dependencies, and Bioconductor build reports, all in \"tidy data\" form. BiocPkgTools can convert from tidy data structures to graph structures, enabling graph-based analytics and visualization. An end-user-friendly graphical package explorer aids in task-centric package discovery. Full documentation and example use cases are included. Availability: The BiocPkgTools software and complete documentation are available from Bioconductor (https://bioconductor.org/packages/BiocPkgTools).", "keywords": [ "bioinformatics", "r", "bioconductor", "software", "reproducible research" ], "content": "Introduction\n\nBioconductor is a open source software project (comprising 1741 individual analysis packages) and community for the analysis and comprehension of large-scale biological data. Newly submitted software packages undergo a technical review to ensure that best practices and Bioconductor coding conventions are followed. The project maintains an automated build system that ensures that packages in the Bioconductor project are compiled and built successfully and pass basic checks. Package downloads are tracked and aggregated by package and month, longitudinally. Finally, package details such as title, description, version, author, and dependencies on other R packages are compiled based on package metadata.\n\nThe current size and growth of the Bioconductor project suggests that there is merit in exposing computable forms of the metadata describing the Bioconductor package ecosystem. To that end, we developed a small suite of tools, BiocPkgTools, to provide easy access to project details such as download statistics, bulk package metadata, and package build status. Developers, project leaders, open source software researchers, and Bioconductor end users can build on the availability of these data for applications such as custom reporting, dependency graph analytics, package filtering, and text mining.\n\n\nFeatures and usage\n\nThe core functionality of BiocPkgTools is to expose Bioconductor project and package metadata as tidy data1 objects (Figure 1). The data presented by the package are accessed directly from online resources available from Bioconductor. As such, the package relies on web connectivity and collects the most recent data. Installation instructions are detailed on the package website.\n\nBiocPkgTools can access and transform web-accessible resources including package metadata, download statistics, dependencies between packages, and updated Bioconductor build report status to \"tidy data\" reports that can be manipulated using standard R tools. Interactive package exploration is also available.\n\nPackage functionality can be roughly divided into data access, data presentation, and graph/network functionality. See Table 1 for an overview.\n\nAfter installing BiocPkgTools, the biocDownloadStats function can generate a tidy data structure summarizing monthly download statistics (both total and unique IP addresses) for all Bioconductor packages.\n\n\n\nThe biocBuildReport function gathers information from the Bioconductor build report site and can be used, for example, to summarize the “build status” for all Bioconductor pacakages.\n\n\n\nThese data are useful to developers to track the health of their software either programmatically or via a searchable, sortable table from the problemPage function.\n\nAs an alternative to basic web browser search and the Bioconductor online software list, the biocExplore function offers interactive and graphical approach to package browsing (see Figure 2). The biocExplore widget allows browsing packages under different biocViews, Bioconductor’s software catergory tags. This interactively visualises the relative number of downloads each package has under different biocViews, allowing users to quickly determine which packages are most commonly used for different analysis tasks.\n\nBubbles are sized based on download statistics. Hovering over a bubble will give download number while clicking on a bubble will pop up a package details page, including a link to the package landing page.\n\nThe Bioconductor package ecosystem is, by design, highly interconnected via package dependencies. Several functions in the BiocPkgTools package provide examples of package dependency graph creation and visualization. Figure 3 displays packages within one degree of dependency relationship of the GEOquery package.\n\nLinks are colored based on type (Suggests [light blue], Depends [green], and Imports [red]) and arrows point to the “dependent” package.\n\nBiocPkgTools is implemented as a standard R package and hosted in the Bioconductor repository. All functions are documented and include examples. An included tutorial (vignette) demonstrates features and capabilities.\n\n\nDiscussion\n\nThe BiocPkgTools package comprises a set of functions for accessing software metadata from the growing collection of Bioconductor packages. For software developers, this metadata can be useful for tracking package build status and the health of package dependencies. Easy access to descriptive package metadata for all Bioconductor software resources can empower researchers or users interested in text mining, custom package search, or analysis of the existing software ecosystem. BiocPkgTools can provide easy access to metrics of Bioconductor sofware usage that are increasingly being incorporated into funding and promotion decisions.\n\n\nData availability\n\nAll data accessed and used by the BiocPkgTools package are publicly available and are updated regularly at the Bioconductor project.\n\n\nSoftware availability\n\nSoftware available from: https://bioconductor.org/packages/BiocPkgTools\n\nSource code available from: https://github.com/seandavi/BiocPkgTools\n\nArchived source code as at time of publication: https://doi.org/doi:10.18129/B9.bioc.BiocPkgTools4\n\nLicense: MIT License", "appendix": "Grant information\n\nResearch reported in this publication was supported by the National Human Genome Research Institute of the National Institutes of Health under award number U41HG004059, the National Cancer Institute of the National Institutes of Health under award numbers U24CA180996 and U01CA214846-02, and the Center for Cancer Research, part of the Intramural Research Program at the National Cancer Institute at the National Institutes of Health. Part of this work was performed on behalf of the SOUND Consortium and funded under the EU H2020 Personalizing Health and Care Program, Action contract number 633974.\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\nWickham H: Tidy data. Journal of Statistical Software, Articles. 2014; 59(10): 1–23. Publisher Full Text\n\nCsardi G, Nepusz T: The igraph software package for complex network research. InterJournal. 2006; Complex Systems, 1695. Reference Source\n\nAlmende B, Thieurmel B, Robert T: visNetwork: Network Visualization using ’vis.js’ Library. R package version 2.0.4. 2018.\n\nSu S, Davis S: BiocPkgTools: Collection of simple tools for learning about Bioc Packages. R package version 1.2.0, 2019. Reference Source" }	[ { "id": "49219", "date": "10 Jun 2019", "name": "Simina M. Boca", "expertise": [ "Reviewer Expertise biostatistics", "bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents the new Bioconductor package, BiocPkgTools, which allows users to obtain various statistics about Bioconductor packages, including the number of downloads by month, the build status, and the package dependencies.\nOverall, this seems to be a very useful package! I'm excited about using it as a way of measuring the use of my own package and perhaps doing some other exploratory analyses on the Bioconductor ecosystem. I was able to load and run everything and obtain similar results to those presented (slight differences due to running it at a different time) after reading this article and the package vignette.\nI believe the following minor edits could improve the presentation:\nProvide the code for generating Figures 2 & 3, even though it is in the vignette. This would be helpful from a reproducibility perspective.\n\nSpecify how often the data presented are updated. Presumably this is done almost immediately, given that the build status is included. It would also be interesting to give an idea of how this is done.\n\nSpecify in the output/help page that the filtering in the bubble chart obtained by running biocExplore() is done by biocViews (this can be a bit confusing since, for example, under \"Bioinformatics\" only a few packages are selected, but that is not something you can fix, since it's up to the authors to include the appropriate biocViews).\n\nSpecify that the stats downloaded from biocDownloadStats() are the total number of downloads, not the number of downloads from distinct IPs, as the Bioconductor package page provides both.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "49235", "date": "19 Jun 2019", "name": "Mike Smith", "expertise": [ "Reviewer Expertise Software development", "Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper Su et al present BiocPkgTools, an R package that provides programmatic access to metadata about software in the Bioconductor project. The package is available from Bioconductor and the source code can be easily viewed on Github. The metadata it can access includes download numbers, package 'health' status, topic categories, and software dependencies.\nSince the software forms at least as large a part of this publication as the article, I have tried to review both.\nPaper: The paper is succinct and to the point. In some instances there is an implied understanding of R package development and the Bioconductor build system. For example, in the code block presented to demonstrate the biocBuildReport function, the row names e.g. buildbin, checksrc etc are probably fairly meaningless to someone unfamiliar with Bioconductor's continuous integration platform. It's fair to say most interested readers will already be Bioconductor package developers, but a caption linking this back to the build system discussion in the introduction would add clarity.\nSimilarly the biocViews concept is not full described, but forms a key part of one the packages main functions: biocExplore(). Expanding a little on how these terms are assigned to packages (i.e. mostly but not always by the package authors) might make it clearer to users why some values return unexpected results e.g. 'Bioinformatics' only shows me 15 packages presumably because most package authors see this as redundant, although 'Software' feels similarly obvious yet returns a huge number of packages as this is assigned by Bioconductor itself.\nThe motivation behind providing programmatic access to build reports and download statistics is presented in the text. However, it would be nice for the authors to expand upon what they feel the use cases for the package dependency graphs may be. They look cool, but based on the paper content it's not immediately obvious to me where they might be used. The discussion highlights the fact that download metrics etc are gaining traction as a measure factored into funding decisions, and one thing that springs to mind is that a similar point can be made visually for software infrastructure that has many downstream dependents. My efforts to create a `subgraphByDegree()` with degree greater than one (to view a larger software stack) creates a graph to large to render, so an example of how to visualise this (i.e. show all downstream dependsOnMe packages in a tree) would be a great addition to either the paper or the package vignette.\n\nMinor points: I recommend providing a link to the BiocPkgTools landing page after the sentence \"Installation instructions are detailed on the package website.\"\nIndicate that the code to produce Figure 3 can be found in the package vignette (or include it as supplementary material here). Since it is quite a bit more than a one-liner to produce Figure 3 I think it would be beneficial to point readers to the code.\nPerhaps I am mistaken, but I can find no mention in the paper or package of what the colours represent in the bubble plots produced by biocExplore(). They definitely feel like they should mean something.\nI would consider rephrasing the sentence \"This interactively visualises the relative number of downloads each package has under different biocViews\". To me this reads like the plot is somehow visualising how relevant the package is to the selected view, when really it is just filtering the package list based on the biocView category.\nTypos (given in context, specific [word] highlighted):\nfor all Bioconductor [pacakages] software [catergory] tags Bioconductor [sofware] usage bioc[b]uildReport (Table 1)\n\nSoftware: In general the software is well written with a comprehensive vignette providing more extensive examples than seen in the paper. However I did encounter a few issues when testing aspects of the package:\nThe function firstInBioc() fails with 'Error in desc(Date) : could not find function \"desc\"'. Similarly, the function inducedSubgraphByPkgs() fails with 'Error in V(g2) <- `vtmp` : could not find function \"V<-\"'\n\nI think in both cases the relevant functions need to be added as imports in the NAMESPACE. These work in the package examples as additional libraries are loaded first.\n\nThe manual page for subgraphByDegree() is slightly confusing. The argument pkg takes a vector of length one, but the description field reads like multiple packages can be supplied. Why is the function get_bioc_data() in snake_case, while all other functions are named with camelCase? I wonder if the function biocBuildEmail() should really be exported. This seems very specific to the Bioconductor core team, but is very similarly named to biocBuildReport() which is one of the primary data getters and will be widely used. Also, the 'Usage' section of the biocBuildEmail function references the function .getTemplatePath() which is not exported, perhaps reflecting the niche nature of this function. It's not clear to me what the difference is between an 'induced subgraph' and a 'regular subgraph' in the functions inducedSubgraphByPkgs() vs subgraphByDegree() and I couldn't find an explanation in the package documentation. Perhaps this is just a lack of knowledge about graphs on my part, but it's information I looked for based on the different function names. Are all of the packages listed in the Suggests field of the DESCRIPTION file used? I can't find mention of tm, SnowballC\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? Yes", "responses": [] }, { "id": "49217", "date": "21 Jun 2019", "name": "Henrik Bengtsson", "expertise": [ "Reviewer Expertise Statistic", "Bioinformatics", "Computer Science", "Scientific Computations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents the BiocPkgTools package, which provides an R API to the various package metadata that is available mostly in human-readable formats on the https://bioconductor.org/ website. By providing an R API for accessing package metadata, the authors argue that \"data mining and value-added functionality such as package searching, text mining, and analytics on packages\" may follow.\nI believe that this package will add value to the Bioconductor community and beyond. It is likely that the package will lower the threshold for doing \"data mining\" on Bioconductor packages. The reports that are based on these metadata and produced by this package are likely to inspire others to produce other types of reports and interactive tools.\nHaving said, I do think there is room for some immediate improvements to the article, which might also spill over to the package itself.\nMajor:\nThe role of the package:\nIt is not clear whether this package is to be considered a Bioconductor core package or a user-contributed package. That can only be inferred/guessed from authors list and possible from the package name.\n\nIf it is an official core Bioconductor package (similar to BiocManager) supported and maintained by the Bioconductor Team, then I think it would be of value to make that explicit. This would change the expectation on the package and its long-term support, e.g. will it break or not when the Bioconductor website changes, what should be documented as part of the package and what should be documented on the Bioconductor website., etc. The questions in the following section illustrate this.\n\nDescription on the metadata:\nThere is no explicit reference to the source data, e.g. is this package using a public or a private Bioconductor Online API to gather the data. Is this API, or URLs official and stable, or is this package meant to play that role?\n\nIs there another reference from where one can learn more about how the data is collected? Are data from other Bioconductor mirrors included in the download stats?\n\nThe download stats do not contain information on the package version, which is mentioned in the package vignette. However, it's not clear whether it is only downloads from the current release branch that are counted or not. For instance, if I download a package for a legacy Bioconductor release version or the current developer version, will that add to the data?\n\nMinor/trivial:\nAPI:\nGiven the package title 'Collection of simple tools for learning about Bioc Packages' and description, the `biocBuildEmail()` function seems to be an odd-one-out.\n\nTypos/spelling:\nSection 'Introduction': \"a open source software\" -> \"an open-source software\".\n\nSection 'Features and usage': missing \"the\" in \"to expose [the] Bioconductor project\".\n\nSection 'Features and usage': \"software cate[r]gory tags\".\n\nSection 'Discussion': ... Bioconductor sof[t]ware...\n\nTable 1: biocbuildReport -> biocBuildReport.\n\nTable 1 caption: \"all Bioconductor pac[a]kages\".\n\nGrammar in Section 'Introduction': Should \"suggest\" be used instead of \"suggests\" in \"The current size and growth of the Bioconductor project suggests\"?\n\nIn addition, `spelling::spell_check_package()` on the package itself reveals several mistakes.\n\nNomenclature: Bioconductor is sometimes referred to as 'bioconductor' (lower case) or just 'bioc' and 'Bioc' (the latter is even used in the package title).\n\nFigures and tables:\nFigure 1: It's not clear what the different parts of this figure are. As I understand it, the left part (the hex logo) represents the package itself, and the middle part (five blocks) represents the \"web-accessible resources\" that the package queries. What does the table and the edges in the right part represent? Is it meant to illustrate that the package pulls data from 5 different sources and joins them into a single table? The caption also tries to mention \"Interactive package exploration is also available\" which makes it harder to understand the figure. Maybe it's the ordering of these three parts are confusing. Maybe it would be more clear if the edges from the hex logo to the web resources would be dropped. Maybe it would help if the empty table could be populated with something else than just \"...\" entries. Alternative, maybe something like this would clarify the flow of the data?\n\nPackage/examples:\nSection 'Implementation': \"All functions are documented and include examples.\" Maybe rephrase so it doesn't sound that there are examples for all functions, which is not the case.\n\nCoding Convention: The package vignette and examples, and the example code snippets in the article, use `a = b` for assignments rather than `a <- b` that is the recommended coding style for Bioconductor, df. https://www.bioconductor.org/developers/how-to/coding-style/.\n\nEXAMPLES: The presented output of the `dlstats = biocDownloadStats()` example is outdated; it shows other fields/columns that what the current BiocPkgTools 1.2.0 outputs.\n\nIn addition to the above, I've identified other package-specific issues that I reported directly to the package's issue tracker (https://github.com/seandavi/BiocPkgTools/issues/34).\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": [] } ]	1	https://f1000research.com/articles/8-752
https://f1000research.com/articles/7-1306/v1	16 Aug 18	{ "type": "Software Tool Article", "title": "An accessible, interactive GenePattern Notebook for analysis and exploration of single-cell transcriptomic data", "authors": [ "Clarence K. Mah", "Thorin Tabor", "Jill P. Mesirov", "Thorin Tabor" ], "abstract": "Single-cell RNA sequencing (scRNA-seq) has emerged as a popular method to profile gene expression at the resolution of individual cells. While there have been methods and software specifically developed to analyze scRNA-seq data, they are most accessible to users who program. We have created a scRNA-seq clustering analysis GenePattern Notebook that provides an interactive, easy-to-use interface for data analysis and exploration of scRNA-Seq data, without the need to write or view any code. The notebook provides a standard scRNA-seq analysis workflow for pre-processing data, identification of sub-populations of cells by clustering, and exploration of biomarkers to characterize heterogeneous cell populations and delineate cell types.", "keywords": [ "scRNA-seq", "single-cell expression", "pre-processing", "clustering", "interactive", "visualization", "GenePattern Notebook", "Jupyter Notebook", "open-source" ], "content": "Introduction\n\nSingle-cell RNA sequencing (scRNA-seq) is a powerful tool to measure genome-wide gene expression at the resolution of individual cells. Compared to traditional RNA-seq collected from bulk cells or tissue, scRNA-seq enables users to capture cell-by-cell transcriptomic variability. This information can then be used to define and characterize heterogeneity within a population of cells, from identifying known cell types to discovering novel ones. A number of high-throughput scRNA-seq protocols have been developed to simultaneously sequence thousands to hundreds of thousands of cells while retaining the origin of each transcript, including SMART-seq2 (Picelli et al., 2014), CEL-seq (Hashimshony et al., 2012), Drop-seq (Macosko et al., 2015), and the commercial 10X Genomics scRNA-seq protocol. Despite the power of this approach, analysis of scRNA-seq data presents a unique set of challenges centered on the discrimination of technical variation from the biological signal. The variability in efficiency of capturing individual transcripts is compounded by the variability in the number of transcripts per cell, anywhere between 50,000 to 300,000 (Marinov et al., 2014). Conversely, reads for multiple cells may be captured together, artificially inflating the number of reads for a single cell. Comprehensive methods and software have been developed for proper data pre-processing, normalization, quality control, and clustering analysis including Seurat (Satija et al., 2015), Scanpy (Wolf et al., 2018), and the 10X Genomics Cell Ranger pipeline. These methods take raw read counts as input and are downstream of read alignment and quantification. They have been used successfully in studies across many cell types to analyze tens of thousands of cells in parallel (Macosko et al., 2015; Svensson et al., 2018; Villani et al., 2017).\n\nWhile these tools are readily available for those with computational expertise who are comfortable programming in Python or R, they are less accessible to non-coding users due to a steep learning curve. In order to enable analysis of scRNA-seq data, regardless of programming expertise, we have created an interactive analysis notebook using the GenePattern Notebook Environment that does not require coding by the user (Reich et al., 2017). The GenePattern Notebook Environment integrates an easy-to-use graphical user interface with the Jupyter notebook's rich text, media, executable code, and results, to present the entire narrative in a single notebook document.\n\nThe notebook presented here aims to provide a standard pre-processing and clustering analysis workflow for scRNA-seq datasets. We based the workflow on the Seurat R tutorial and perform the below analysis steps using methods implemented in the Scanpy Python package.\n\n\nMethods\n\nThe workflow begins with an expression data matrix already derived from alignment of reads and quantification of RNA transcripts. Each row of the matrix should represent a gene and each column represents a cell. Gene by cell matrices generated by the 10X Genomics Cell Ranger pipeline and flat text files from read count quantification tools like HTSeq (Anders et al., 2015) and kallisto (Bray et al., 2016) are supported as input.\n\nOnce the expression matrix is loaded into the notebook using a GenePattern cell (Figure 1A), the notebook presents a series of plots to compare quality metrics across cells (Figure 1B). There are 3 metrics including: the number of genes detected in each cell, the total counts in each cell, and the percentage of counts mapped to mitochondrial genes. A high percentage of mitochondrial genes indicates the cell may have lysed before isolation, losing cytoplasmic RNA and retaining RNA enclosed in the mitochondria. The user can interactively set thresholds to see how the number of cells below the threshold change (Figure 1B).\n\n(A) The “Setup Analysis” function is presented using the GenePattern UI Builder. (B) The quality metric distributions are shown as kernel density estimation fitted curves. The values of the mean, 3 standard deviations (SDs) above the mean and 4 SDs above the mean are indicated to help identify outlier cells with abnormally large metric values. Interactive sliders under each plot allow the user to see how many cells are included under a threshold.\n\nWe encourage the user to visually inspect their data across several parameters, using the quality metric plots provided prior to proceeding with further analysis. Furthermore, we enable the user to determine appropriate filtering thresholds for each of the metrics to exclude low quality cells and outliers by inputting thresholds in the GenePattern cell interface (Figure 2A). We have also provided an option to filter for genes expressed in a minimum number of cells. All preprocessing steps follow the Seurat and Scanpy workflows. Counts are scaled to have the same total counts for each cell. Highly variable genes are identified for downstream analysis by selecting genes with a minimum mean expression and dispersion; where dispersion is calculated as the log of the mean to variance ratio. Counts are then log-transformed to reduce the distribution skew and bring it closer to a normal distribution. To remove sources of technical variation, linear regression is used to diminish the effects of the number of detected molecules and the percentage of counts mapped to mitochondrial genes. Finally, the counts for highly variable genes in each cell are scaled to unit variance and a mean of zero. For clustering cells in the next step, dimensionality reduction is performed using principal component analysis (PCA) on highly variable genes. A plot showing the standard deviation of each principal component is then displayed so the user may choose a reasonable number of principal components for use in clustering (Figure 2B).\n\n(A) The “Preprocess Counts” function is presented using the GenePattern UI Builder. Here the user specifies thresholds for filtering samples and for performing log normalization. (B) A scatterplot showing the percent variance explained by each individual principal component.\n\nAs suggested in Satija et al., 2015, and followed in the Seurat and Scanpy workflows, we cluster cells using a graph-based clustering approach. With the selected principal components as features, the cells are embedded in a K-nearest neighbor graph where cells are grouped using the Louvain community detection method (Blondel et al., 2008). Then t-distributed stochastic neighbor embedding (t-SNE), a standard dimensionality reduction technique suited for visualizing high-dimensional data, is used to project and visualize the cells in the space of the first two t-SNE components (Figure 3) (Maaten & Hinton, 2008). Cells are represented as points colored by clustering assignment. Select parameters including the number of principal components, Louvain clustering resolution, and t-SNE perplexity are exposed for users to iteratively adjust the clustering results using the visualization for feedback (Figure 3). Setting a higher resolution results in more and smaller clusters. The perplexity parameter loosely models the number of close neighbors each cell will have.\n\nThe clustering parameters can be changed using the sliders and re-plotted with the “Plot” button. Cells are projected into t-SNE space, with the first two t-SNE components as the axes of the plot. Cluster assignments of cells are defined by Louvain clustering and denoted as distinct colors.\n\nThe application of proper visualization tools is an important aid to interpret the complexity and depth of scRNA-seq data. We provide various visualizations within the notebook to explore differentially expressed genes, which can be used to identify specific cell types or highlight heterogeneous gene expression across clusters (Figure 4A and B, Figure 5). There is also an interface to query for differentially expressed genes that are higher in one cluster compared to the rest (Figure 4C). The Wilcoxon-Rank-Sum test statistic is used to rank genes by default. This test is performed in a one-versus-all setup for each of the clusters, providing unique markers for each individual cluster. We also include the option to perform pairwise cluster comparisons. Additional statistical information about each gene is provided as an interactive table, such as the log-fold change comparing the average expression of a gene in one cluster versus the average expression in all other cells, the percentage of cells within the cluster that express the gene, and the percentage of cells in other clusters that express the gene. The percent expression metric shows whether a reasonable fraction of cells expresses the gene.\n\n(A) A heatmap showing the expression of the top 10 differentially expressed markers of each cluster across all cells. (B) A violin plot illustrating the distribution of expression of the gene MS4A1 in each cluster. Expression of MS4A1 is a canonical marker of B cells, which clearly has a positive distribution higher expression in cluster 2 compared to the other clusters. (C) An interface to perform differential expression between one cluster versus the rest. Results are shown in the table.\n\nCells are projected into t-SNE space, as in Figure 2, but are colored by the relative expression of a given gene instead of cluster assignment. Colors span a gradient from red (high expression) to grey (low expression). Genes shown here are indicative of known cell types; MS4A1: B cells, GNLY: NK cells, CD3E: T cells, CD14: CD14+ monocytes, FCER1A: dendritic cells, FCGR3A: FCGR3A+ monocytes, LYZ: CD14+ monocytes, PPBP: megakaryocytes, and CD8A: CD8 T cells.\n\nData generated by the analysis can be exported in two ways. First, as a set of CSV (comma separated values) files suited for further independent analysis and data sharing. We provide a description of the exported CSV files, which include the preprocessed expression matrix, cell annotations, dimensional reduction outputs, and gene rankings generated during the analysis. Second, as an H5AD file that can be re-imported into this notebook’s workflow, retaining the generated results. The parameters for each step in the analyses are automatically saved in the notebook once executed, ensuring the entire workflow is documented. Notably, the entire notebook can be shared with other users rather than exporting output files.\n\nTo run the notebook, the user is required to have a GenePattern account that can be created on the GenePattern Notebook site. After logging in, the notebook can be found in the “Featured” section of the “Public Notebooks” page.\n\n\nUse case\n\nAn example notebook (https://github.com/genepattern/single_cell_clustering_notebook) employs a scRNA-seq gene expression dataset for 2700 peripheral blood mononuclear cells (PBMCs) from a healthy donor as a demonstration of its use. We can recapitulate cell types identified using Seurat and Scanpy; the clusters can be characterized by visualizing the expression of canonical markers of these cell types on the 2D t-SNE projection plot. We also find that many of these markers are highly ranked when looking at significant differentially expressed genes between clusters (Figure 4). In Figure 4 we examine cluster markers to understand why some larger groups of cells are divided into sub clusters.\n\nFor example, LYZ is overexpressed in a cloud of samples that clustering separates as two distinct clusters, 1 and 5. The LYZ gene encodes for human lysozyme, an antimicrobial agent associated with blood monocytes. Using the cluster comparison tool (Figure 4C), we can see that cluster 1 exhibits high relative expression of CD14 while cluster 5 exhibits high relative expression of FCGR3A, also known as the CD16 receptor gene (Figure 5). These two genes characterize two known subtypes of blood monocytes respectively; classical and non-classical monocytes.\n\n\nConclusion\n\nAs single-cell RNA-seq continues to grow in popularity, this GenePattern Notebook will provide an accessible and reproducible way to preprocess the data and perform clustering analysis without having to interact with any code. We plan to continually review the notebook as single-cell RNA-seq protocols evolve to be even more high-throughput and as algorithms adapt to accommodate growing amounts of single-cell data. As the GenePattern Notebook user interface gains more features, the notebook will also grow to take advantage of these features. Future notebooks such as those for multi-experiment aggregation (multiple sequencing runs) and pseudotime analysis are being considered to grow a compendium of single-cell sequencing analysis notebooks.\n\n\nSoftware and data availability\n\nGenePattern Notebook is available from: https://genepattern-notebook.org/.\n\nGenePattern Notebook source code is available from: https://github.com/genepattern/seurat_python_notebook.\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.1326656 (Mah, 2018).\n\nLicense: BSD 3-Clause\n\nThe 3k PBMCs from a Healthy Donor dataset is publicly available via the 10X Genomics website after user registration: https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNIH U24CA194107, NIH U41HG007517, NIH U19AI090023, Silicon Valley Community Foundation 2018-183110 (5022).\n\nAll funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors thank Nadia Arang, Olivier Harismendy, Lukas Chavez and members of the Mesirov Lab for providing feedback on the workflow and manuscript, and the GenePattern development team for help implementing features in the GenePattern Notebook. The authors also thank Dan Carlin, Konstantin Okonechnikov, Alexander Wenzel, and Edwin Juarez for testing the notebook on independent data sets.\n\n\nReferences\n\nAnders S, Pyl PT, Huber W: HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31(2): 166–169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlondel VD, Guillaume JL, Lambiotte R, et al.: Fast unfolding of communities in large networks. Journal of Statistical Mechanics: Theory and Experiment. 2008; 2008(10): P10008. Publisher Full Text\n\nBray NL, Pimentel H, Melsted P, et al.: Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016; 34(5): 525–527. PubMed Abstract \| Publisher Full Text\n\nHashimshony T, Wagner F, Sher N, et al.: CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell Rep. 2012; 2(3): 666–673. PubMed Abstract \| Publisher Full Text\n\nMaaten LJP, Hinton GE: Visualizing High-Dimensional Data Using t-SNE. J Mach Learn Res. 2008; 9: 2579–2605. Reference Source\n\nMacosko EZ, Basu A, Satija R, et al.: Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015; 161(5): 1202–1214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMah C: genepattern/single_cell_clustering_notebook: v1.0.2 (Version v1.0.2). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1328256\n\nMarinov GK, Williams BA, McCue K, et al.: From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing. Genome Res. 2014; 24(3): 496–510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPicelli S, Faridani OR, Björklund ÅK, et al.: Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014; 9(1): 171–181. PubMed Abstract \| Publisher Full Text\n\nReich M, Tabor T, Liefeld T, et al.: The GenePattern Notebook Environment. Cell Syst. 2017; 5(2): 149–151.e1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSatija R, Farrell JA, Gennert D, et al.: Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015; 33(5): 495–502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSvensson V, Teichmann SA, Stegle O: SpatialDE: identification of spatially variable genes. Nat Methods. 2018; 15(5): 343–346. PubMed Abstract \| Publisher Full Text\n\nVillani AC, Satija R, Reynolds G, et al.: Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017; 356(6335): pii: eaah4573. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolf FA, Angerer P, Theis FJ: SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018; 19(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "37288", "date": "17 Sep 2018", "name": "Timothy Tickle", "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\n“An accessible, interactive GenePattern Notebook for analysis and exploration of single-cell transcriptomic data” by Mah et al announces GenePattern NoteBooks to provide an interactive, easy-to-use interface for data analysis and exploration of single cell transcriptomics data.\nGenePattern is an important body of software that supports an active user base. Growing resources for single cell transcriptomics will continue to enable that community and extend GenePattern to additional users. Jupyter notebooks are an excellent tool for development and reproducibility of scripts, it is interesting to see it being leveraged as a space to provide user interfaces for single cell transcriptomics data science. The selection of analysis was well selected, using a tutorial from Seurat, a leading environment for single cell transcriptomics analysis who have historically provided a collection of high-quality tutorials. Steps for analysis leveraged from that tutorial seem reasonable and visualizations (excluding one comment below) seem to match what you would see in publications.\nThe following major comments should be addressed.\nThe current manuscript focuses on user experience through a standardized analysis pattern, this is done well by the publication. The majority of the analysis and data visualization is modeled after current trends but two areas of improvement exist. The use of a Wilcoxon-Rank-Sum test to test one label verse all other labels is useful to researchers and is something offered through current analysis packages. There has been work that has improved the way differential expression is performed, SCDE and MAST are packages that allow more complex models or comparisons that go beyond two labels. Of those, at least MAST has shown to be performant and is now available as an option in Seurat. It is rare one will only find two clusters of cells in a single cell transcriptomics study, the extension to more labels should be included. Heatmaps were used in Figure 4a. Although useful when one wants to see the actual data and scalable to an extremely small subsection of genes (here 10), often single cell transcriptomics data is too sparse to fully appreciate patterns when presented in heatmaps (as measurements, when not using summaries) and the numbers of observations in these studies can go to more than a million; making this visualization not scalable. Dot plots should be the first plot offered in these use cases with the ability to go to heatmaps to see the actual data if needed. Standardization and approachable user experiences are big wins for our community, but this has to be coupled with an underlying methodology that is scalable to the sizes of data we see and expect to see the near future. Data sets of over a million already exist, how does this solution scale to data in the thousands, tens of thousands, and so on. It is essential the manuscript include benchmarking so users can understand if working with their data set in this environment is possible. Please list if there are any costs (or that there are no costs if that is, in fact, true) with running analysis in the notebooks. Must one always download the notebooks and run them on their own systems or is the running of analysis hosted?\n\nA response to following minor comments are of interest to the reviewer.\nIt is interesting that Jupyter Notebooks are leveraged to target an audience that can not program given Jupyter notebooks are a common environment for developers. That being said the interfaces given to the users do seem to be appropriate user experiences for those who prefer working through UIs. Does this work also include the ability for someone to edit and update the code of the GenePattern Notebook if they are a developer? Such a functionality would extend the usability of the notebooks by supporting an additional type of user. In Figure 1 kernel density estimations with a data plotted below the density are used instead of violin plots with overplotted data. Although I appreciate the same information is being presented in both plots (hence this being a minor comment), it would be helpful to use violin plots instead of the current plots. First, violin plots are de facto (and the GenePattern plot does not add information) and secondly, outliers are given more presence in violin plots (data is plotted directly on the plot and the tail of the plot to higher values is not hidden by an axis). This is important given outlier are explicitly the focus of figure 1.\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": "4315", "date": "18 Dec 2018", "name": "Jill Mesirov", "role": "Author Response", "response": "We appreciate your comprehensive comments and suggestions for improvement and enhancement of the notebook and are working to incorporate them as well as revise the paper. When that work is finished we will provide a more detailed response to review." }, { "c_id": "4537", "date": "29 May 2019", "name": "Clarence Mah", "role": "Author Response", "response": "1. Data I/O. I attempted to use the web-hosted version of the notebook to upload a large csv (80 MB). After nothing happened, I switched to a smaller CSV. Since it was in gene x cell format instead of cell x gene, the analysis couldn't proceed. Finally I tried a 10X .mtx file, but as there was no way to upload the corresponding barcodes file, that didn't work. Finally I used an h5ad file from a previous scanpy analysis. That won't be possible for the typical user. (Given slow upload times, actually, you may want to accept zipped csvs as well.)The author should make it possible to upload 10X files properly. They should also allow for the data to be gene x cell or cell x gene, by giving the user a chance to transpose the matrix if necessary.We have addressed the lack of flexibility in regards to input files and mention that in the text.Users can now upload or link to their own 10X files (matrix, genes, and barcodes) or upload a single matrix file and specify whether it contains “gene by cell” or “cell by gene” data. Any of these files may be zipped as well. The new text reads as follows:“The workflow begins with an expression data matrix already derived from alignment of reads and quantification of RNA transcripts. Users may upload a single expression file and specify whether the rows represent genes and the columns represent cells or vice-versa. Text files from read count quantification tools like HTSeq (Anders et al. 2015) and Kallisto (Bray et al. 2016) are supported as input. Additionally, this notebook supports the three-file 10X output format, allowing users to upload the matrix, genes, and barcodes files. Any of those inputs can also be provided as .zip files.”We thank the reviewer for providing additional files for us to use in testing and we have confirmed that the notebook will load even the larger 80MB file.2. Interactive sliders are nice, much better than setting cutoffs by number, seeing how values change, and iterating. For the cutoffs on nGene and nCounts, the sliders are not aligned with the plot and lack numerical axes or numbers for current values. These should be displayed directly under the plots so that one can slide them to align with suggested cutoffs at various values of sigma, and should also display the numerical value currently selected.(Also, the suggestion that 3 is an appropriate cutoff is based on the assumption of a normal distribution, which does not hold for this data, especially for 10X.)We have adjusted the alignment of plot elements such that the sliders are now more closely aligned with the boundaries of their respective KDE plots. We have left the graphical markers indicating the third and fourth standard deviation on the plots for the purpose of describing the distribution, but we have removed the suggestion that those values may make good cutoffs.3. The notebook bakes in certain analysis choices, such as regressing out % mito, which are not statistically sound. For the problems with regressing out, see this [blog post](http://ds.czbiohub.org/blog/Regression-Hazards/). I suggest that such \"corrections\" be removed. For a simple, sound analysis, see the workflow and language we used for Tabula Muris [Annotation Vignette](https://github.com/czbiohub/tabula-muris/blob/master/00_data_ingest/02_tissue_analysis_rmd/Organ_Annotation_Vignette.Rmd)We appreciate the reviewer's word of caution with regards to regressing out %mito and the links to some relevant literature. We included some of these analysis choices as we are explicitly following the Seurat tutorial, and that is a data preprocessing step in the tutorial. However, we added a note of caution to the text of the manuscript and the GenePattern notebook with regards to the possible hazards of regression in the single cell RNA-seq context while noting the alternatives provided, and we have made the regression step optional. The text now reads as follows:We also give users the option to use remove sources of technical variation by performing linear regression on the total number of molecules detected and the percentage of reads mapped to mitochondrial genes. As there is debate in the field concerning the correctness of using regression on covariates such as percent mitochondrial reads (Batson 2018) we have made this step optional.4. The notebook also includes some assumptions about the reference used. In particular, it assumes for % mito that genes be formatted in a certain case.For the purposes of reproducing the Seurat tutorial, we kept the convention of assuming that mitochondrial gene names begin with “MT-”. This “hardcoding” could be avoided by asking users to supply a list of mitochondrial genes corresponding to their data’s gene ID system, but we feel this might over-complicate the input step and impose a burden on users, especially since, as the reviewer observes, the percent mitochondrial genes regression is of questionable statistical utility in the general case. We have emphasized in the manuscript that gene names must follow this specific convention in order to use this regression. Because of this requirement as well as the statistical issues the reviewer notes above, we have made this regression step optional. Users who either do not wish to use regression or do not have gene names in the required format can now skip this step.5. Users will likely have metadata they want to visualize, such as sample, batch, sex, stimulated vs unstimulated, etc. They should be able to upload a csv with that data, and visualize it on the tSNE plots. This is important for interpretation of the results.We agree with the reviewer that this is an important step in single cell RNA-seq workflows. However, an appropriate implementation of visualization of metadata is a feature that we believe would require a separate notebook and is out of scope of the goal of this particular notebook. Thus, we leave this for future work.6. The tSNE tab for visualizing gene expression did not load when clicked on.We have identified the source of this error as an incompatibility between GenePattern Notebooks and certain Jupyter Widgets (ipywidgets) that arose after the initial release of this notebook. We are addressing this problem; however, in order to avoid further delays we have redesigned the visualization without using the incompatible widgets. Correspondingly, Figure 4 in the manuscript has been updated to reflect this change.7. Conversely, reads for multiple cells may be captured together, artificially inflating the number of reads for a single cell. \" Doublet detection is indeed a tricky problem. There are [methods](https://www.biorxiv.org/content/early/2018/07/19/352484) to address it, but this notebook does not implement them.We appreciate the importance of considering problems such as doublet detection. The Seurat PBMC tutorial does not include a doublet detection step and Scanpy does not currently implement methods for handling doublets. However, we do mention the importance of this issue in the text and point to a reference, and that we hope to address this issue in future work. The relevant text reads as follows:For example, future notebook releases may include quality control methods such as doublet detection (McGinnis et al., 2018) as well as visualization methods such as UMAP (Becht et al., 2019), which is growing in popularity in the single cell community.8. Depending on the size of the data, each step may take seconds to hours. For a naive user, they may not know how long to wait and at some point anyone would give up. It would be very useful if the author did some calibration for each step (running sample datasets of various sizes) so that an estimated time to completion could be displayed.We have annotated each step in the notebook with benchmarking for the Seurat PBMC dataset. For each step, we list the default amount for the limiting factors (genes, principal components, etc.) and the execution time we estimate for that step based on those values. For example, “For 2,600 cells and 10 principal components, this step will run in approximately 60 to 90 seconds”. Additionally, the Scanpy developers have benchmarked their code both on the same Seurat PBMC dataset we use in this notebook and on an large dataset of one million cells. We have cited these benchmarks in the manuscript in the first paragraph of the conclusions.Final remarks:Single-cell sequencing analysis is evolving, and it is essential that we get the most sound methods in the hands of practitioners. Anchoring this notebook to Scanpy is great, since that library is actively being developed. I recommend that the author regularly update this template with methods as they become available in that library. For example, t-tests and the wilcox have problems with log-normalized data (they fail to be consistent when cells are sampled to different depths). I recommend the t-test_overestim_var from Scanpy for something fast and logreg for something more accurate, but all the options should be made available and the defaults from Scanpy should be the defaults for you.This will need to be a living document to be useful. If it is a repository of best practices for simple single-cell analysis, then it may serve as a way in to single for people who don't yet know how to program.We agree with the reviewer that keeping this notebook in line with evolving best practices is essential. We also agree that Scanpy’s default parameters are likely robust in the general case, however, in keeping with the objective of following the Seurat PBMC tutorial, default settings for parameters in this notebook were chosen to reproduce the results from that tutorial. We have included documentation at each step in the notebook to introduce users to the purpose and nuance of different parameters and empower them to make the best choices for their own data." } ] }, { "id": "40612", "date": "15 Nov 2018", "name": "Joshua Batson", "expertise": [ "Reviewer Expertise computational analysis of biomedical data", "including single-cell sequencing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 GenePattern Notebook implementing a standard analysis pipeline for single-cell RNA sequencing data. GenePattern notebooks allow a user to access python libraries for data analysis through a simple GUI--dropdown menus, text fields, and sliders are used to provide inputs to python functions that get run under the hood. This allows a user familiar with web interfaces to run a program without having to understand syntax.\nIn this case, that program is Scanpy, a package for scRNA-seq analysis. The order of the steps and the supporting guidelines for parameter choice and data exploration are taken from the Seurat PBMC3k tutorial.\nIdeally, a naive user could upload their gene counts (say the output of CellRanger by 10X), tune just a few parameters according to the guidelines in the notebook, and find meaningful clusters in their data. Such an accessible analysis with guard-rails would be useful--had I been aware of the GenePattern system when organizing data analysis for the Tabula Muris project, I would certainly have used it, and so avoided many problems with people deleting code they shouldn't have, not changing file names to match their local directory structure, etc.\nThere are some significant usability issues with the notebook as it stands that will need to be remedied for it to be useful to a broad audience. Since people who cannot program will be unable to make even small changes to the workflow, it is important that the pipeline implemented here by scientifically sound, robust to different input formats and scales, and feature-complete.\nData I/O. I attempted to use the web-hosted version of the notebook to upload a large csv (80 MB). After nothing happened, I switched to a smaller CSV. Since it was in gene x cell format instead of cell x gene, the analysis couldn't proceed. Finally I tried a 10X .mtx file, but as there was no way to upload the corresponding barcodes file, that didn't work. Finally I used an h5ad file from a previous scanpy analysis. That won't be possible for the typical user. (Given slow upload times, actually, you may want to accept zipped csvs as well.)\nThe author should make it possible to upload 10X files properly. They should also allow for the data to be gene x cell or cell x gene, by giving the user a chance to transpose the matrix if necessary.\nInteractive sliders are nice, much better than setting cutoffs by number, seeing how values change, and iterating. For the cutoffs on nGene and nCounts, the sliders are not aligned with the plot and lack numerical axes or numbers for current values. These should be displayed directly under the plots so that one can slide them to align with suggested cutoffs at various values of sigma, and should also display the numerical value currently selected.\n\n(Also, the suggestion that 3\\sigma is an appropriate cutoff is based on the assumption of a normal distribution, which does not hold for this data, especially for 10X.)\nThe notebook bakes in certain analysis choices, such as regressing out % mito, which are not statistically sound. For the problems with regressing out, see this [blog post](http://ds.czbiohub.org/blog/Regression-Hazards/). I suggest that such \"corrections\" be removed. For a simple, sound analysis, see the workflow and language we used for Tabula Muris [Annotation Vignette](https://github.com/czbiohub/tabula-muris/blob/master/00_data_ingest/02_tissue_analysis_rmd/Organ_Annotation_Vignette.Rmd) The notebook also includes some assumptions about the reference used. In particular, it assumes for % mito that genes be formatted in a certain case. Users will likely have metadata they want to visualize, such as sample, batch, sex, stimulated vs unstimulated, etc. They should be able to upload a csv with that data, and visualize it on the tSNE plots. This is important for interpretation of the results. The tSNE tab for visualizing gene expression did not load when clicked on. \"Conversely, reads for multiple cells may be captured together, artificially inflating the number of reads for a single cell. \" Doublet detection is indeed a tricky problem. There are [methods](https://www.biorxiv.org/content/early/2018/07/19/352484) to address it, but this notebook does not implement them. Depending on the size of the data, each step may take seconds to hours. For a naive user, they may not know how long to wait and at some point anyone would give up. It would be very useful if the author did some calibration for each step (running sample datasets of various sizes) so that an estimated time to completion could be displayed.\nFinal remarks:\nSingle-cell sequencing analysis is evolving, and it is essential that we get the most sound methods in the hands of practitioners. Anchoring this notebook to Scanpy is great, since that library is actively being developed. I recommend that the author regularly update this template with methods as they become available in that library. For example, t-tests and the wilcox have problems with log-normalized data (they fail to be consistent when cells are sampled to different depths). I recommend the t-test_overestim_var from Scanpy for something fast and logreg for something more accurate, but all the options should be made available and the defaults from Scanpy should be the defaults for you.\nThis will need to be a living document to be useful. If it is a repository of best practices for simple single-cell analysis, then it may serve as a way in to single for people who don't yet know how to program.\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": "4314", "date": "18 Dec 2018", "name": "Jill Mesirov", "role": "Author Response", "response": "We appreciate your comprehensive comments and suggestions for improvement and enhancement of the notebook and are working to incorporate them as well as revise the paper. When that work is finished we will provide a more detailed response to review." }, { "c_id": "4538", "date": "29 May 2019", "name": "Clarence Mah", "role": "Author Response", "response": "1. The current manuscript focuses on user experience through a standardized analysis pattern, this is done well by the publication. The majority of the analysis and data visualization is modeled after current trends but two areas of improvement exist. The use of a Wilcoxon-Rank-Sum test to test one label verse all other labels is useful to researchers and is something offered through current analysis packages. There has been work that has improved the way differential expression is performed, SCDE and MAST are packages that allow more complex models or comparisons that go beyond two labels. Of those, at least MAST has shown to be performant and is now available as an option in Seurat. It is rare one will only find two clusters of cells in a single cell transcriptomics study, the extension to more labels should be included. We agree with the reviewer that other analyses, for example more complex differential expression, can be performed. However, writing suitable Python wrappers for those is beyond the scope of this article and notebook. As Scanpy is constantly updated to reflect these current best practices to analyze single cell RNA-Seq data, this notebook can be updated as well. 2. Heatmaps were used in Figure 4a. Although useful when one wants to see the actual data and scalable to an extremely small subsection of genes (here 10), often single cell transcriptomics data is too sparse to fully appreciate patterns when presented in heatmaps (as measurements, when not using summaries) and the numbers of observations in these studies can go to more than a million; making this visualization not scalable. Dot plots should be the first plot offered in these use cases with the ability to go to heatmaps to see the actual data if needed. We first note that, due to software compatibility issues with visualization widgets, we have redesigned the presentation of Figure 4 in the manuscript and the visualization in Step 4 of the notebook. However, we have chosen, in this case, to continue displaying the heatmap first. We agree with the reviewer that heatmaps for large, sparse data are useful mostly for summarizing, and that is indeed the reason we provide this heatmap. Users can identify the top n markers of each cluster in the heatmap (now Step 4A) and then explore each marker in more detail using the tSNE and violin plots in Step 4B. 3. Standardization and approachable user experiences are big wins for our community, but this has to be coupled with an underlying methodology that is scalable to the sizes of data we see and expect to see the near future. Data sets of over a million already exist, how does this solution scale to data in the thousands, tens of thousands, and so on. It is essential the manuscript include benchmarking so users can understand if working with their data set in this environment is possible. We have included benchmarking relevant to the Seurat PBMC tutorial dataset before each step in the notebook. Additionally, the Scanpy development team has benchmarked their code (which forms the core of the analysis performed by this notebook) on both the same Seurat tutorial dataset we use in the notebook and a large dataset of one million cells. We have cited these benchmarks in the manuscript (see response to Reviewer 1). We note that the GenePattern Notebook uses a client-server architecture with most processing done by the server. Most datasets on the order of thousands of cells can likely be processed using the free public GenePattern Notebook server at https://notebook.genepattern.org. For larger datasets, the open source GenePattern Notebook server can be deployed on any appropriate computational resource, for example, an Amazon AWS instance or an institutional supercomputer allocation. We have added text to the manuscript noting this – see point 4 below. 4. Please list if there are any costs (or that there are no costs if that is, in fact, true) with running analysis in the notebooks. Must one always download the notebooks and run them on their own systems or is the running of analysis hosted? We have added the following text to the conclusion section of the manuscript to address this concern: We encourage users to perform analyses on their own data using this notebook. We note that all the required libraries are already installed on the public GenePattern Notebook server at https://notebook.genepattern.org. This resource is freely available to the community and the analysis described in this notebook falls well within the per-account memory allocations (see the Scanpy authors’ benchmarking in Wolf et al., 2018, Eulenberg et al., 2017, Eulenberg et al. 2, 2017). To analyze larger datasets that exceed the per-user memory allocation on the public notebook server, users should deploy the open source GenePattern Notebook server using their own computational resources as described in Reich et al., 2017. The GenePattern Notebook server is available as the genepattern-notebook package through the pip (https://pypi.org/project/genepattern-notebook/) or conda (https://anaconda.org/genepattern/genepattern-notebook) package managers, or as a Docker image (https://hub.docker.com/r/genepattern/genepattern-notebook). A response to following minor comments are of interest to the reviewer. 1. It is interesting that Jupyter Notebooks are leveraged to target an audience that can not program given Jupyter notebooks are a common environment for developers. That being said the interfaces given to the users do seem to be appropriate user experiences for those who prefer working through UIs. Does this work also include the ability for someone to edit and update the code of the GenePattern Notebook if they are a developer? Such a functionality would extend the usability of the notebooks by supporting an additional type of user. Yes, GenePattern Notebooks allow users who are comfortable programming to modify the underlying code or add their own. Users access public GenePattern notebooks by saving a copy of the notebook to their own accounts. In addition to executing the analysis with the notebook copy, users can modify their copy, share it with other users, and publish modified versions to a notebook repository. Please refer to the article describing GenePattern Notebook for further details (https://doi.org/10.1016/j.cels.2017.07.003) or to the section on Programmatic Features of the GenePattern Notebook website (http://genepattern-notebook.org/programmatic/). 2. In Figure 1 kernel density estimations with a data plotted below the density are used instead of violin plots with over-plotted data. Although I appreciate the same information is being presented in both plots (hence this being a minor comment), it would be helpful to use violin plots instead of the current plots. First, violin plots are de facto (and the GenePattern plot does not add information) and secondly, outliers are given more presence in violin plots (data is plotted directly on the plot and the tail of the plot to higher values is not hidden by an axis). This is important given outlier are explicitly the focus of figure 1. In Figure 1, cells are displayed under the density plots (in the form of dots) with outliers clearly visible. We believe this already implemented functionality achieves the effect of highlighting the outliers in the distribution as requested by the reviewer." } ] } ]	1	https://f1000research.com/articles/7-1306
https://f1000research.com/articles/8-746/v1	28 May 19	{ "type": "Software Tool Article", "title": "A new method for detecting abrupt shifts in time series", "authors": [ "Chris A. Boulton", "Timothy M. Lenton", "Timothy M. Lenton" ], "abstract": "Abrupt shifts in time series are a topic of growing interest in a number of research areas. They can be caused by a range of different underlying dynamics, for example, via a mathematical bifurcation, or potentially as the result of an auto-correlated stochastic process (i.e. ‘red’ noise). Here we present a method that detects abrupt shifts by searching for gradient changes that occur over a short space of time. It can be automated, allowing many time series to be analysed by the user at once, such as from high spatial resolution data. Our method detects abrupt shifts regardless of their origin (which it cannot deduce). We present a comparison with the method of abrupt shift detection from the changepoint R package, which is based on changes in mean over the time series. Our method performs better on data with an underlying trend where comparisons of means may fail.", "keywords": [ "Abrupt shifts", "Time series analysis", "Shift detection", "R package", "Methods" ], "content": "Introduction\n\nAbrupt shifts are found in time series in many fields, including but not limited to ecology1,2, climate3–6, medicine7 and engineering8. Abrupt shifts in time series can be caused by a range of different underlying dynamics. They are sometimes described as ‘tipping points’3,6 and are caused by an underlying mathematical bifurcation9, implying some change in control parameters governing the system in question. Alternatively, noise-induced transitions can occur between different attractors, triggered purely by stochastic variability. Sufficiently rapid forcing could also trigger an abrupt response from a system without passing a bifurcation10. In other situations, a strongly auto-correlated stochastic process (‘red’ noise) can produce what appear as abrupt shifts in time series, although it is debated whether they should be described as such11.\n\nOur method is based on the presumption that when people search for abrupt shifts in time series by eye, they are looking for significant changes in the gradient of the time series over a short space of time. Hence we have designed a method based on looking for anomalous rates of change in a time series and released it as an R package called ‘asdetect’12 There exist several other methods of detecting anomalous changes in time series (change-point analysis13,14, STARS15, etc) which are based on looking for significant shifts in the distribution of the data. These attempt to fit generalised models to the data, or use t-tests (or other variants) to detect a significant shift in the distribution of segments of the time series. In contrast, our algorithm focuses more on the transition itself rather than the resulting behaviour of the time series (i.e. differences between the situation before and after). Thus, the approaches can be viewed as complementary. Our method should be intuitive to users, particularly those with little statistics experience. It is also useful for analysing a number of time series at once when it would be otherwise impossible to view them all individually.\n\nFigure 1 provides a graphical representation of how our method differs from the widely-used ‘changepoint’ analysis package from R16. This method is used in the function as_detect() in our R package. For a time series that exhibits a typical abrupt shift that users might be searching for (Figure 1a), both methods detect a shift. For a drifting time series, however, a change in mean is detected from the change-point algorithm but an abrupt shift is not detected by our method (Figure 1b). These example time series, particularly the time series that exhibits the abrupt shift are used throughout the article.\n\nThe tool looks for changes in the gradient of the time series (blue) compared to the changepoint analysis R package that looks for changes in mean value over time (red). (a) An example time series that undergoes a bifurcation and hence there is an abrupt shift. (b) A drifting time series where a change in the mean is detected by the changepoint algorithm but this does not relate to an abrupt shift. These example time series are discussed in more detail later.\n\nOur algorithm searches for anomalous changes in the gradient of a time series. We begin with a time series of length n (an example is shown in Figure 2), which we separate into l-sized segments. Across each segment, we fit a linear regression and record the gradient/coefficient of each of the n/l segments. For ls that are not factors of n and thus would give a remainder, we spread these remaining points equally at the start and end, such that our segments would focus on the centre of the time series rather than at the beginning or end.\n\nThree choices of l, 25 (a–c), 50 (d–f), and 100 (g–i) are shown. Full explanation of the plots is given in the main text. In (a), (d) and (g), the example time series is shown in grey with red lines showing the linear regressions fitted through each section. In (b), (e) and (h), the gradients of each linear regression are shown, along with dotted lines representing the 3 median absolute deviations (MADs). Panels (c), (f) and (i) show the evolving detection time series, which is described in detail in the main text.\n\nWe look for which of these gradients are significantly different by seeing which values lie outside ± 3 median absolute deviations (MADs) of the distribution of gradients. For those points in time that contribute to a significant gradient, we add or (subtract for significantly lower/negative gradients) 1 to the relevant l-long segment(s) of a ‘detection time series’ of length n which contains only 0s otherwise.\n\nThis process is repeated over a range of ls. The minimum and maximum l is chosen by the user (although both have a default; see below), and the process is repeated for all integers between these values. The resulting detection time series is divided by the number of ls tested, which gives a time series showing the (sign-included) proportion of times that a point contributed to a significant gradient. From this, a user-defined threshold (magnitude) can be used to determine if an abrupt shift is detected in the time series.\n\nOur second function, where_as(), takes the detection time series as well as this threshold magnitude (between 0 and 1) to locate where the abrupt shifts are detected. The function records the indices where the detection time series is beyond the threshold magnitude, using the sign of the detection times series to distinguish increasing or decreasing abrupt shifts. The R function rle(), which computes the lengths of runs of equal values in a vector, is used on the differences of the list of indices that are above the threshold. This allows us to determine the sections of the time series that contain the detected abrupt shift(s). Within these subsets, we then find the maximum or minimum (depending on whether the detection is positive or negative) value to report as the position of an abrupt shift.\n\nWhile it is easy to find a single maximum/minimum in the detection time series, our second function allows any number of abrupt shifts to be found, depending on how strict the user wants to be. If no values in the detection time series are found to be above the threshold level, our function will issue a warning and instead return the maximum detected value and position.\n\n\nMethods\n\nOur R package asdetect12 contains a number of functions which allows the user to detect abrupt shifts in either their own data, or in example time series created by functions within the package.\n\nOur algorithm in as_detect() begins by separating a time series of length n into l-sized segments. Within each segment, we fit a linear regression of the form Y = a + bX where Y is the values in the time series, X is the vector, [1⋮l], b is the gradient of the regression model (which is recorded) and a is the intercept of the model (estimate of Y when X=0). For the set of gradients, we calculate how many are more than 3 median absolute deviations (MADs) away from the median of the set of gradients, where 1 MAD = median(\|x-median(x)\|). MADs are a substitute for standard deviations, which could be heavily influenced by large outliers. Points in the time series that contribute to significant gradients have a value of 1 added or subtracted to a detection time series, which is then divided by the number of l used.\n\nThe model for bifurcating example time series (shown in Figure 1–Figure 4) is:\n\n\n\n(a) The example time series. (b) Detection time series is shown in black where it is above the threshold value (0.7, vertical dotted line; as described in the main text) and grey otherwise. Vertical dotted lines denote the range where the detection time series is above the threshold (778-872) whereas the solid lines denote the maximum detection value (0.99) and position (838).\n\n(a, b) Abrupt shift (as in Figure 1 and Figure 2), (c,d) white noise, (e,f) a drifting time series, and (g,h) the PDO Index. Details of the detections are described in the main text.\n\nWhere x is the time series, µ is a parameter that causes the bifurcation. It runs linearly from 0 to 239 at t=900, up to t=1000. Stochastic white noise is applied to the system (ση), sampled from a normal distribution with mean 0 and standard deviation σ which we set to 0.1. We start the time series at x=-1, the stable state when μ=0. This time series can be recreated with the function tip_eg() in our R package.\n\nOur example time series of white noise and drifting white noise has the same level of stochastic forcing as the bifurcation time series above, centred on -1:\n\n\n\nWith the same parameter choices as in Equation (1). The value of D is set to 0 for our white noise time series, and ranges from 0 to 2 across the 1000 points of the drifting time series seen later. A similar time series, centred on 0 rather than -1 can be created from our function white_noise_eg() in our R package.\n\nThe flat start and end time series are created from combinations of the white noise and drifting white noise time series, using the D parameter. For example, for the flat start time series, D is set to 0 for the first 200 points and then increases at the same rate as previously, such that it runs linearly from 0 to 1.6 over the over 800 time points. The time series for the flat end, D runs from 0 to 1.6 over the first 800 time points, and then remains at 1.6 for the final 200 time points.\n\nOur Pacific Decadal Oscillation (PDO) Index example is taken from the Joint Institute for the Study of the Atmosphere and Ocean, and is the first principal component of sea surface temperatures in the North Pacific once an annual cycle and general warming trend have been removed. This time series is not included within our package.\n\nWe use the cpt.mean() function of the R package ‘changepoint’, with its default settings as described in the help file of the function.\n\nOur package is designed to be used in standard R and has no dependencies on other packages apart from installation. As asdetect12 is hosted on GitHub, users will first need to download the ‘devtools’ package, using the command install.packages(‘devtools’) in the R console. Then our package can be installed using the command devtools::install_github(‘caboulton/asdetect’) and then loaded with the command library(‘asdetect’).\n\n\nResults\n\nFigure 2 shows an example of how the detection time series is built up, over three different l choices, for an example time series of data. A similar time series can be created with our tip_eg(), as described in the Methods. We note, as mentioned above, the full algorithm uses all integer values between the range prescribed, but only show three distinct values as a demonstration of how our method works. The model underlying the example time series exhibits a bifurcation at t=900; however, the system is forced with stochastic noise which causes the system to tip somewhat earlier. We stress that our algorithm is ignorant of how the shift in the time series occurs, but such a canonical example makes it clearer to illustrate its use.\n\nWe begin by using a value of l of 25 (Figure 2a) and fit the linear regressions through each 25 length segments (red lines; Figure 2a). There are two significant gradients found with l=25 (Figure 2b), a significantly negative gradient for the points 801–825 and a significantly positive gradient for points 826–850. This results in -1 and 1 values stored in the detection time series in Figure 2c. We then imagine we are next running the analysis using a value of 50 for l (Figure 2d), although in reality the value of l increases by 1 each time. This time there are significant positive gradients found for points 751–850 (Figure 2e), which counteracts the negatives found with l=25. This causes the points 801–825 to revert back to 0. We also divide the detection time series through by 2 as we have tried for both l=25 and 50 (Figure 2f). This results in a peak of 1 (gradients significantly positive for both values of l) for points 826–850, 0.5 for points 751–800 (those were only significantly positive for l=50) and 0 for points 801–825 (those which were significantly negative for l=25, and positive for l=50), as well as the 0s elsewhere. Finally, we again run the analysis using l=100 (Figure 2g). We find a significantly positive gradient for 801–900 (Figure 2h), leading to a detection algorithm that contains 1s at points 826–850, with 0.33 assigned to the other points between 751–900 (Figure 2i).\n\nRunning the full algorithm, for all values of l between 5 and 300 (rather than just 3 specific choices of l as shown in Figure 2), for the example time series (Figure 3a), we use the resulting detection time series in our second function (Figure 3b). We use a threshold of 0.7 and find that points 778–872 are above this threshold. Within this, we find the highest detection (0.993) at point 838 (Figure 3b). Point 838 in the example time series is the first point the time series is positive and as such is a good indicator that for this particular underlying system the time series has switched from one (disappearing) basin of attraction to the other.\n\nFigure 4 shows that in contrast to the time series with an underlying bifurcation (Figure 4a, b), a time series of constant white noise (Figure 4c), has a maximum absolute detection value of (-0.115) and point 794 (Figure 4d). We also examine a time series of drifting white noise (Figure 4e), which shows a maximum detection value of 0.12 at point 518 (Figure 4f). These detection values from the other time series are much lower than those found in the time series with underlying bifurcation and would not be detected by our second algorithm (with only the maximum and a warning returned). We use the PDO index as a 4th example (Figure 4g). This is the major mode of climate variability in the North Pacific once the annual cycle and overall warning trend have been removed17. There is debate in the literature about whether or not there are abrupt shifts in the index, linked to changes in climate regime18,19, or if it is actually a red noise process that drifts above and below a single mean state11. This real-world time series acts as an interesting case study for our algorithm. Our method detects no significant abrupt shift (Figure 4h) with a maximum absolute detection value of -0.108 in May 1942. This suggests that, at least according to our method, that the PDO is a red noise process, as no shifts are detected.\n\nA caveat of our algorithm is that it detects all anomalous gradients some of which would not be described as abrupt changes. For example, where a time series is relatively constant for a short period at the start before a longer period of increasing, then the short period at the start will be detected as anomalous (an unusually low gradient relative to the rest of the time series). This is also true if a short flat section is detected at the end of an increasing or decreasing time series. Examples of these cases are shown in Figure 5. Figure 5 also shows that these types of behaviour are not necessarily picked up strongly, with minimum detection values of -0.69 and -0.39 for increasing and decreasing time series, respectively. Similar behaviour is also detected for flat sections in otherwise decreasing time series. These are detected as increasing abrupt shifts (not shown).\n\nFlat sections occur at the (a, b) start or (c, d) end of increasing time series are detected as decreasing abrupt shifts as their gradients are significantly lower than the rest of the time series.\n\nThe example time series (Figure 1 and Figure 2) is tested when using a threshold of (a) 0.7 (as in Figure 2), (b) 0.88, and (c) 0.95. A second smaller shift is detected with the middle threshold (refer to Figure 1 and Figure 2 to link this back to the original time series).\n\nWe have created a third function, shift_type(), that checks if the abrupt shifts that are detected within time series are ‘flat’ or not. True abrupt shifts that most users will be searching for will have stronger gradients closer to the detected position than the gradient of the time series overall. Our third function compares the absolute value of the gradient of a linear regression model fitted locally around the detected position compared to the absolute gradient of a linear regression fitted across the full time series. We treat those shifts with smaller gradients compared to the overall gradient as ‘flat’. The subset of the time series the local linear regression model is fitted to is a user defined choice, but we recommend (as is the default) it to be 10% of the total length of the time series, 5% either side. If a shift is detected within the first or last 5% (or within the length determined by the user), then the function will use data up to the start or end of the time series, even if this results in a smaller window that the model is fitted to.\n\nTo determine how our algorithm compares to the changepoint package, we compare the detections in six example time series, the tipping point time series, white noise, drifting white noise, and we also include two time series with flat sections at the beginning and end of otherwise increasing time series, which provides an example of what is described above. Analyses of the PDO index are also compared.\n\nTable 1 shows the positions of abrupt shifts detected by both methods, including the detection value from our algorithm and the confidence value from changepoint. We also include the actual position that the abrupt shift is in the time series. In changepoint we use the cpt.mean() function. cpt.mean() searches for changes in the mean across the time series, and we use the ‘AMOC’ method such that a maximum of one will be detected. The position returned is the beginning of the second section that has a significantly different mean to the first.\n\nResults in brackets are not detected as shifts by our algorithm, or deemed significant by the changepoint package.\n\n*Although the bifurcation in the tipping point time series happens at 900, the noise pushes it to an abrupt shifts before this (as described in the main text).\n\nWe find that both methods agree exactly with where the abrupt shift occurs in the tipping point time series. There are no abrupt shifts detected in the white noise series, with a detection value of -0.115 only being picked up if the user used such a low threshold for detection. The same is true of our algorithm for the drifting time series. However, the changepoint algorithm detects a change at point 520 (with confidence 0.917). For the flat time series, the detections from our algorithm are much closer to the actual change in the time series, although the detection values are relatively low. This is useful as it is not the type of abrupt shift that would usually want to be detected but is still highlighted. These types of anomalies are also picked up by our third function. Changepoint detects changes about half way through the time series, with much higher confidence values. This is a simple result of the changepoint method looking for changes in the mean.\n\nWhile changepoint manages to detect the tipping point time series as well as our own method does, it struggles with time series where there is no abrupt shift but an overall trend. It is also noticeable that the confidence values are a lot higher than our detection values in the ‘flat’ time series, despite being further away from the actual transition. However, we iterate that the changepoint function is looking for changes in the mean over the time series and so although is used for detecting abrupt shifts, there is no differentiating between fast and slow shifts. It detects a shift in the drifting time series as that is what it is designed to do. Our method, however, which focuses on gradients, finds no abrupt shift.\n\n\nDiscussion\n\nThe main inputs of our algorithm are the choice of minimum and maximum l. This depends on the length of the time series such that significant gradients can still be found. A significant gradient can only be found if there are three or more segments and thus the maximum value of l should be less than 1/3 of n, the length of the time series. Any choice of l above this will cause the maximum possible detection value to decrease as well as all the detection values to be lower than they otherwise would be. Having a choice of maximum l too low, however, may bias detection towards short term fluctuations in the time series rather than the type of abrupt shift-type behaviour that was intended. As a default of our function, a maximum value of l equal to 1/3 of n is used.\n\nA choice for the minimum value is more difficult to determine. It is required to be big enough such that if a time series is ‘noisy’, then it will not detect significant gradient changes due to deviations caused by low-level noise. Less noisy (smoother) systems can have a smaller minimum value, and as such will pick up smaller abrupt shifts which would be difficult in a time series with more noise. We set a default of 5 points as a ‘rule of thumb’ for being able to find a significant coefficient in a linear regression model.\n\nAnother choice of variable is the threshold in our second function to determine where abrupt shifts are detected. Within a consecutive set of points, the function will search for the maximum value within this range. However, it does not take into consideration local maxima (or minima) that are still above the threshold. This is a design choice of our algorithm, as we assume that users will want to only find the largest abrupt shift locally, and that the smaller nearby shifts (local maxima/minima) have had their detection values inflated due to being near the large abrupt shift.\n\nAn example is found in our original case (Figure 2). We showed using a threshold of 0.7 that an abrupt shift was detected (Figure 3). However, using a threshold value of 0.88, we find another abrupt shift at point 800 (detection is 0.91), as well as the original at point 838 (detection: 0.99). This can be seen as a large excursion that (just) failed to leave the disappearing basin of attraction, in this case which is approaching an underlying bifurcation. Figure 6 shows that the local minimum in the detection time series is below the threshold and thus a new segment is detected. A threshold value of 0.95, again only detects the original shift.\n\nWe have outlined our method for detecting abrupt shifts in time series, which is based on looking for significant changes in gradient of the series. Our algorithms differ from those such as the changepoint R package, which looks for significant changes in mean values. We have shown that this method detects shifts in time series that drift when they would not ordinary be deemed abrupt shifts by users. Our alternative method does not suffer from this set-back. Furthermore, it is intuitive in its design, whilst allowing greater control for users to discover abrupt shifts of different magnitudes.\n\n\nData availability\n\nThe PDO Index time series can be found at the Joint Institute for the Study of the Atmosphere and Ocean website.\n\nOther, stochastic, time series were created using our package. To create a time series that contains an abrupt shift, the function tip_eg() is used. An example of white noise with or without drift can be created with the white_noise_eg(). Time series that began or ended with flat sections used a combination of two white_noise_eg() outputs that were stitched together to give the desired outcome.\n\n\nSoftware availability\n\nSource code available from: https://github.com/caboulton/asdetect/.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.263404412.\n\nLicense: MIT License.", "appendix": "Grant information\n\nThis work was funded by the NERC Valuing Nature programme, grant number NE/P007880/1.\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\nScheffer M, Bascompte J, Brock WA, et al.: Early-warning signals for critical transitions. Nature. 2009; 461(7260): 53–9. PubMed Abstract \| Publisher Full Text\n\nLitzow MA, Urban JD, Laurel BJ: Increased spatial variance accompanies reorganization of two continental shelf ecosystems. Ecol Appl. 2008; 18(6): 1331–1337. PubMed Abstract \| Publisher Full Text\n\nLenton TM, Held H, Kriegler E, et al.: Tipping elements in the Earth's climate system. Proc Natl Acad Sci U S A. 2008; 105(6): 1786–1793. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeld H, Kleinen T: Detection of climate system bifurcations by degenerate fingerprinting. Geophys Res Lett. 2004; 31(23). Publisher Full Text\n\nDakos V, Scheffer M, van Nes EH, et al.: Slowing down as an early warning signal for abrupt climate change. Proc Natl Acad Sci U S A. 2008; 105(38): 14308–14312. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLenton TM: Early warning of climate tipping points. Nat Clim Chang. 2011; 1: 201–209. Publisher Full Text\n\nScheffer M, Bolhuis JE, Borsboom D, et al.: Quantifying resilience of humans and other animals. Proc Natl Acad Sci U S A. 2018; 115(47): 11883–11890. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLivina V, Barton E, Forbes A: Tipping point analysis of the NPL footbridge. Journal of Civil Structural Health Monitoring. 2014; 4(2): 91–98. Publisher Full Text\n\nThompson JMT, Sieber J: Predicting climate tipping as a noisy bifurcation: a review. Int J Bifurcat Chaos. 2011; 21(02): 399–423. Publisher Full Text\n\nAshwin P, Wieczorek S, Vitolo, R, et al.: Tipping points in open systems: bifurcation, noise-induced and rate-dependent examples in the climate system. Philos Trans A Math Phys Eng Sci. 2012; 370(1962): 1166–1184. PubMed Abstract \| Publisher Full Text\n\nRudnick DL, Davis RE: Red noise and regime shifts. Deep Sea Research Part I: Oceanographic Research Papers. 2003; 50(6): 691–699. Publisher Full Text\n\nBoulton CA: asdetect: An R package for detecting abrupt shifts in time series. 2019. http://www.doi.org/10.5281/zenodo.2634045\n\nBeaulieu C, Sarmiento JL, Mikaloff Fletcher SE, et al.: Identification and characterization of abrupt changes in the land uptake of carbon. Global Biogeochem Cycles. 2012; 26. Publisher Full Text\n\nBeaulieu C, Chen J, Sarmiento JL: Change-point analysis as a tool to detect abrupt climate variations. Philos Trans A Math Phys Eng Sci. 2012; 370(1962): 1228–1249. PubMed Abstract \| Publisher Full Text\n\nRodionov SN: A sequential algorithm for testing climate regime shifts. Geophys Res Lett. 2004; 31. Publisher Full Text\n\nKillick R, Eckley IA: changepoint: An R Package for Changepoint Analysis. J Stat Softw. 2014; 58(3): 19. Publisher Full Text\n\nMantua NJ, Hare SR, Zhang Y, et al.: A Pacific Interdecadal Climate Oscillation with Impacts on Salmon Production. B Am Meteorol Soc. 1997; 78: 1069–1080. Publisher Full Text\n\nHare SR, Mantua NJ: Empirical evidence for North Pacific regime shifts in 1977 and 1989. Prog Oceanogr. 2000; 47(2–4): 103–145. Publisher Full Text\n\nScheffer M, Carpenter S, Foley JA, et al.: Catastrophic shifts in ecosystems. Nature. 2001; 413(6856): 591–596. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49169", "date": "20 Jun 2019", "name": "Valerie N. Livina", "expertise": [ "Reviewer Expertise 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\nThe paper by Boulton and Lenton “A new method for detecting abrupt shifts in time series” proposes an alternative to the previously published change-point techniques (methodology and R package software).\nI think the paper is interesting and can be indexed after revision. In particular, the authors may be interested to look at the recent paper: Beaulieu C. and R. Killick (2018)1 and the corresponding software package ‘EnvCpt’ that performs automatic selection between a variety of trends, changepoints and autocorrelation models, found here.\n\nOn page 2, the authors mention that the red noise “can produce what appears as abrupt shift in time series, although it is debated whether they should be described as such”. A time series with a dynamically changing scaling exponent of red noise actually provides the underlying mechanism of a gradual tipping, as confirmed by several indicators for early warning signals (see Prettyman et al., EPL 20182). This should be distinguished from the red noise with a single fixed scaling exponent that does not change with time (and that may be the case of absence of a tipping point). This needs to be discussed more clearly, otherwise an unprepared reader could be misled regarding the role of memory in tipping dynamics. In particular, this should be better described in the Abstract (or removed from there for simplicity).\nSince the object of analysis is a single-variable time series, the authors actually calculate not gradient (which is by definition multi-variate) but a discrete derivative in each subset of data.\nIt would be good to see a systematic ensemble experiment estimating the success rate of the technique performance for a range of: jumps heights at change points (with respect to the series noise level), lengths of time series, and window sizes (similar to Fig.2 but in a broader range of parameters and with summary over multiple samples rather than a few examples).\nIt is not clear to me why the detection series is always within the interval [-1,1] – do the authors normalise this series? On page 2, the authors say that they “add or subtract 1 to the relevant l-long segments of a detection time series which contains only 0s otherwise”- but in (Fig.2f,i) there are also non-zero and non-one values, why?\nThe discussion of the threshold choice could be improved for better clarity, in my opinion. Maybe some diagram could help visually here.\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": "49168", "date": "03 Jul 2019", "name": "Sonia Kéfi", "expertise": [ "Reviewer Expertise Ecology", "theoretical ecology", "alternative stable states", "tipping points", "facilitation", "ecological networks." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors present a method and the R-package 'asdetect' to detect abrupt shifts in time series data. The approach relies on identifying anomalous changes in the gradient of a time series.\nThe tools provided by the R-package 'asdetect' are important and interesting. They will be a nice complement to the set of tools already available in the literature. For example, previous approaches such as one of the functions implemented in the R package 'changepoint' searches for changes in the mean, rather than the gradient.\nThe paper is well-written. The methods presentation could nonetheless be improved. I hereafter suggest some changes that may help improve the clarity and make the paper more accessible.\nI would slightly reorganize the structure of the paper to gather all the methods in a single place in the paper. As it is now, part of the methods is in the introduction and the rest in the methods. Part of the information needed to understand the methods is also online, in the help of the R-package and in the Readme file of the github repository. It would greatly improve the clarity of the methods presentation if all the information needed was gathered in a single location. Therefore, I would move the last 5 paragraphs of the introduction to the methods part (all the text after 'We begin with a time series of length n', at the bottom of the left column of p. 2). In the third paragraph of the left column of p. 2, I would also move the sentence mentioning the function 'as_detect()' to the methods. This will make the introduction less technical and more focused on giving the intuition of the methods. This will also decrease the amount of repetitions between the introduction and the methods.\n\nIn the introduction, after the third paragraph of the left column of p. 2, I would spend a few more words explaining the intuition of the approach and the difference with other methods, such as the ones implemented in the 'changepoint' package. Figure 1b is a convincing illustration of the differences in outputs given by the two approaches for drifting time series. However, there is also not quite enough information about 'changepoint' for the reader to truly understands what it does (and why, in the example of Figure 1b, it detects a shift in the middle of the time series). I am not very familiar with 'changepoint', but it seems that the package provides several methods to detect changes in time series and that cpt.mean(), which is the function used by the authors for the comparison here, is only one of them. As mentioned by the authors, there are also other methods and packages available and it's unclear why so much emphasis is put on 'changepoint' in this paper. I would clarify all of this in the introduction, and in particular state that Figure 1b is one illustrative example of a striking difference between this approach and previous ones, obtained when using one of the possible functions of 'changepoint'.\n\nThe presentation of the methods could be improved by more explicitly mentioning the different steps of the analysis. - Merging the end of the introduction with the start of the 'Implementation' part of the methods will help. - Also, a graphical presentation of the different steps performed with the names of the corresponding functions of the package as well as what they take as input and what they give as output would be very useful. - When the function as_detect() is mentioned in the text, it would help to say that is takes a time series as input and gives back a 'detection time series' as output. - The 'detection time series' should be clearly defined the first time it is mentioned (i.e. the fact that it is a vector of the same length of the time series which will contains information about the proportion of times a significant gradient was detected at that time step). Right now, there is a vague explanation in the first paragraph of the right column of p. 2 ('For those points in time...'), but the actual explanation is in the results part of the paper. It could stay that way, but then the first time it is mentioned, instead of a general reference to Figure 2, a more specific reference to the left column of Figure 2 would be less confusing, because the reader only gets the information needed to understand the two other columns of Figure 2 (and especially how panels f and i are obtained) after reading the elements currently given in the methods. An alternative may be to move the explanation of Figure 2 to the methods. - Also, it might be helpful to mention in the legend of Figure 2 that panel f contains the information from b and e, and that panel i is derived from information from b, e and h. - In the second paragraph of the right column of p. 2, \"although both have a default; see below\". I would specify that this is in the discussion of the paper (e.g. replace 'see below' by 'see discussion'). - At the beginning of the last paragraph of p. 2, when the function where_as() is introduced, it would help to mention the two outputs of that function. - It was not really clear to me what the function rle() gives as an output (last paragraph of p. 2). It would also help if such information was added to the readme file of the Github repository (which is really useful to get practically started with the package). - In the methods, I would divide the 'implementation' part in two: one part explaining the approach (and including the last paragraphs of the current introduction), and another part for the presentation of the example time series used (i.e. in the right column of p. 3 create a new subparts called 'Data'?).\n\nIn the results, in the second paragraph of p. 6, I did not find the explanations of what happens for the flat time series very clear. Why is this 'useful as it is not the type of abrupt shift that would usually want to be detected'? I am not sure what the authors mean. How is this also 'picked up by the third function'? (Here, I would also mention the name of the function explicitly.) - Caveats of the approach are presented in the results of the flat sections of time series. Is that the only situation where this approach is expected to give a false positive? Are there other situations where this method would fail to detect a shift or wrongly detect one?\n\nThe authors briefly mention in the introduction of the paper that this approach can be viewed as complementary to others. I would mention that again in the discussion by saying something about the difficulty in detecting shifts in noisy data and the importance of using a combination of tools to improve detectability.\nMinor points:\nIn the abstract, it is mentioned that 'we present a comparison with the method...': this comparison is however not really systematic and complete, in particular since (as far as I understood) it uses only one of the functions of the package. I would reformulate this sentence e.g. as 'we illustrate differences with one of the commonly used function of the changepoint R package'.\n\nIn the readme file of the Github repository, it would help to add that after installation, the package has to be loaded with library('asdetect'), as it is done in the text in the 'Operation' part.\n\nIn the help of the as_detect() function, the default value for lowwl is not mentioned(?).\n\nIn the results, first paragraph, the sentence 'a similar time series...' can be deleted. There is no need to repeat this.\n\nIn the results, first paragraph, '..., but we only show three distinct values' ('we' lacking?).\n\nIn the results, second paragraph, 'This results in -1 and 1': I would remind at the end of that sentence that the detection time series has the same length as the original time series.\n\nIn the results, second paragraph of p. 4: 'in our second function': I would say the name of the function for clarity.\n\nFirst paragraph of p. 5: 'according to our method, the PDO is a red noise process' (remove 'that'?).\n\nSecond paragraph of the left column of p. 5: I was a bit confused by the sentence: '...these types of behaviour are not necessarily picked up strongly...' but the value of -0.69 is pretty high, isn't?\n\nSecond paragraph of the left column of p. 5, last sentence: maybe show this?\n\nLast paragraph of the left column of p. 5: 'We have created a third function...', but other functions have been mentioned in between. To avoid confusion, I would say 'we have created another function...'. Also, it would be helpful to add this function (with an example of use) in the readme file of the Github repository.\n\nLast paragraph of p. 5: maybe specify again that you use only one of the functions of change point (maybe if this is more clearly stated at the beginning of the paper, this won't be needed here anymore).\n\nLast paragraph of the results: '...and so although it is used for detecting...' ('it' missing?)\n\nFirst paragraph of the right column of p. 6: the maximum value of l is mentioned two times in that paragraph. Delete one of them.\n\nThird paragraph of the right column of p. 6: 'our second function': for clarity, mention the name of the function.\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": [] } ]	1	https://f1000research.com/articles/8-746
https://f1000research.com/articles/8-175/v1	11 Feb 19	{ "type": "Research Article", "title": "Health and saliva microbiomes of a semi-urbanized indigenous tribe in Peninsular Malaysia", "authors": [ "Li-Fang Yeo", "Farhang F. Aghakhanian", "James S. Y. Tan", "Han Ming Gan", "Maude E. Phipps", "Li-Fang Yeo", "Farhang F. Aghakhanian", "James S. Y. Tan", "Han Ming Gan" ], "abstract": "Background: The indigenous people of Peninsular Malaysia, also known as Orang Asli, have gradually been urbanized. A shift towards non-communicable diseases commonly associated with sedentary lifestyles have been reported in many tribes. This study engaged with a semi-urbanized Temiar tribe from Kampong Pos Piah, Perak, who are experiencing an epidemiological transition. Methods: Weight, height, waist circumference, blood pressure, HbA1C and lipid levels were measured as indicators of cardio-metabolic health. DNA was extracted from saliva using salting-out method followed by PCR amplification of the V3-V4 region of the 16S rRNA gene and sequencing on Illumina MiSeq. Microbiome analysis was conducted on Qiime v1.9. Statistical analysis was conducted using Qiime v1.9 and R.\n\nResults: The study revealed that 60.4% of the Temiar community were overweight/obese, with a higher prevalence among women. HbA1C levels showed that 45% of Temiar had pre-diabetes. Insulin resistance was identified in 21% of Temiar by using a surrogate marker, TG/HDL. In total, 56.5% of Temiar were pre-hypertensive, and the condition was prevalent across all age-groups. The saliva microbiome profiles of Temiar revealed significant differences by gender, BMI, abdominal obesity as well as smoking status. The relative abundance of Bifidobacterium, bacteria commonly found in dairy products, was increased in men. Prevotella, associated with consumption of plant-rich diets, was increased in women. Mogibacteriacea and Mycoplasma levels were significantly elevated in overweight individuals. Proteobacteria was significantly depleted in smokers. Conclusions: Temiar from Pos Piah had a high prevalence of cardio-metabolic risks, including general and abdominal obesity, pre-diabetes, prehypertension and hypertension. This phenomenon has not been previously reported in this tribe. The saliva microbiome profiles were significantly different for individuals of different gender, BMI scores and abdominal obesity and smoking status.", "keywords": [ "Orang Asli", "saliva microbiome", "anthropometrics", "cardio-metabolic health", "indigenous people" ], "content": "Introduction\n\nThe Orang Asli (OA), which means “original people” in the Malay language, comprise approximately 0.5% (150,000) of the total Malaysian population1. They are categorized into three main groups, namely Negrito, Senoi and Proto Malay. OAs are widely spread across the Peninsular and range from semi-nomadic deep forest hunter-gatherers such as the Jahai to resettled communities such as Mah Meri to urbanized city-fringe dwellers such as Orang Seletar1. This study focused on the Temiar who are a subtribe of Senoi and are believed to be descendants of the first Neolithic farmers who migrated to the Malay Peninsula2.\n\nIn recent years, many OA communities were resettled by the government in effort to improve their lives. As the OA became more urbanized and by large left their ancestral habitats and practices, they led more sedentary lifestyles. These factors, coupled with loss of access to forest resources and increasing pressures to turn to store-bought food, may largely explain the rise in cardio-metabolic diseases such as hypertension, diabetes and obesity shown in recent studies1,3,4.\n\nThe launch of the Human Microbiome Project heralded the unprecedented investigations of various microbiomes. Of these, oral microbiomes had been widely studied in human health and diseases5,6. Studies implied an oral origin to systemic diseases such as cardiovascular diseases as the oral cavity is a major gateway into the body7,8. Studies also investigated associations between oral microbiome and diabetes9,10 and obesity10, with mixed results. There were suggestions that obese people may have a different salivary bacterial composition perhaps akin to inflammation, which contributed to periodontal diseases and caries11.\n\nLittle is known about the microbiomes of indigenous communities in Asia. To our knowledge, this was a pioneering investigation of their saliva microbiomes. Furthermore, biomedical studies of Temiar were sparse and outdated, despite them being a very large community. With this impetus, our study aimed to address the gap in knowledge by reporting on the anthropometrics and cardio-metabolic health of a resettled Temiar community and investigated their saliva microbiome in association with their health.\n\n\nResults\n\nA total of 72 Temiars, 33 men and 39 women, participated in the study. The median age was 34 years old. General and abdominal obesity had higher prevalence among Temiar women (Table 1). Notably, 71.4% (n=25) of women and 28.1% (n=9) of men displayed abdominal obesity.\n\nHbA1C levels indicated 44.9% of Temiar to be pre-diabetic, with a higher prevalence in men. The high prevalence of prediabetes is worrying because it indicates a rise in non-communicable diseases that was previously of low prevalence in rural communities. Using a TG/HDL as a surrogate marker for insulin resistance, 22% of Temiar were at risk of IR, mostly affecting men.\n\nBlood pressure measurements showed that 56.5% (n=39) had pre-hypertension, which was more prevalent among women and was prevalent across all age groups. Stage 1 hypertension prevalence rate was 17.4% (n=12) and was found more prevalent among men. Raw data for these measurements are available in figshare12.\n\nTo analyse the saliva microbiota diversity, the V3-V4 hypervariable region on the 16S rRNA gene was amplified and sequenced on Illumina MiSeq. After data quality control (QC), a total of 991,006 reads with mean 14,362±78 reads per individual remained.\n\nTo investigate whether the samples were sequenced to a sufficient depth, a rarefaction curve was plotted using the alpha diversity metric, Phylogenetic Distance. The plateau showed in the rarefaction curve indicated that all 69 samples were sequenced to a sufficient depth (Figure 1). Reads were aligned to Greengenes database V13. The major phyla observed include Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria (Figure 2). These are the features of common oral microbiomes13.\n\nPERMANOVA was used to investigate the saliva microbiome compositions and were found to be associated with gender, waist circumference and smoking habits. To determine whether the relative abundance of individual bacterial species was differently represented in association with the factors investigated, we used the Kruskal-Wallis test on both weighted and unweighted UniFrac distance metrices. UniFrac14 is a phylogeny-based distance matrix that takes into account the relatedness between each bacterial species. Unweighted UniFrac is a qualitative metric that considers only the presence or absence of the bacteria whereas weighted UniFrac is a quantitative metric that considers the abundance of the bacteria.\n\nWeighted UniFrac revealed that the dominant bacterial species present in the saliva were not significantly different among gender (p-value = 0.165). However, we found that the saliva microbiomes differed significantly between men and women for unweighted UniFrac (p-value = 0.028, Figure 3a). Kruskal-Wallis test revealed that the relative abundance of Prevotella, Capnocytophaga, Leptotrichia, Neisseria and Streptococcus were significantly increased in women’s saliva microbiomes (Table 2). These commensal oral bacteria may become opportunistic pathogens in immune-compromised states. The relative abundance of Bifidobacterium, the ‘good bacteria’ commonly found in dairy products, was found to be highly elevated in men.\n\nPCoA plots with the larger shapes representing the mean of distance matrix for (a) gender; (b) body mass index (BMI); (c) smoking status.\n\nThe saliva microbiome profiles also differed significantly with BMI (weighted UniFrac, p-value = 0.01; unweighted UniFrac, p-value = 0.029, Figure 3b). A post-hoc Dunn’s test that conducted pairwise comparisons between underweight, normal and overweight groups revealed that the oral microbiome profiles of underweight individuals differed significantly from both overweight and normal individuals (underweight vs normal group, p-value = 0.0179; underweight vs overweight group, p-value = 0.0007). There was no significant difference between the saliva microbiome profiles of normal and overweight individuals (p-value = 0.0819). Use of the Kruskal-Wallis test using R revealed higher relative abundance of Mogibacteriaceae and Mycoplasma in the microbiomes of overweight individuals (FDR q-value = 0.035, FDR q-value = 0.035). The Kruskal-Wallis test generated by QIIME revealed no significant difference.\n\nThere was a significant difference between waist circumference and saliva microbiome composition (weighted UniFrac, p-value = 0.022). However, there was no significant difference in unweighted UniFrac (p-value = 0.286) as well as in individual bacterial taxa (p-value>0.05) among healthy individuals and those with abdominal obesity. The saliva microbiome composition of non-diabetic, pre-diabetic and diabetic individuals suggested some differences, but they were not significant (unweighted p-value = 0.069, weighted p-value = 0.122). The saliva microbiome composition and relative abundance of specific bacterial species did not statistically differ by age group, lipid levels nor blood pressure levels.\n\nThere was a perceptible difference in the saliva microbiomes and smoking habits using weighted UniFrac (p-value = 0.016, Figure 3c) but no difference detected when using unweighted UniFrac on QIIME (p-value = 0.059). Further testing showed the relative abundance of Proteobacteria and Firmicutes (phylum level) were significantly different among smokers and non-smokers. Within the phylum Proteobacteria, the relative abundance of Neisseria and Aggregatibacter was decreased in smokers compared to never-smokers and former smokers. Current smokers had a lower abundance of Neisseria and Aggregatibacter than former smokers, but the difference was not statistically significant (p-value>0.05). The relative abundance of Campylobacter and bacteria of the class Clostridia, under the phylum Firmicutes were higher in both current and former smokers compared to never-smokers (Table 3).\n\nOverall, the relative abundance of Neisseria and Aggregatibacter was decreased in current and former smokers, whereas the relative abundance of Campylobacter and Clostridia was greater in smokers. The saliva microbiome showed no significant difference between former smokers and never-smokers, suggesting perhaps the saliva microbiome may recover partially to an environment prior to smoking. Relative abundance of bacteria such as Neisseria, Aggregatibacter and Camplyobacter was most affected in smokers, followed by former smokers.\n\n\nDiscussion\n\nWe reported a high prevalence of cardio-metabolic diseases such as obesity, pre-diabetes, insulin resistance and pre-hypertension among Temiar. These non-communicable diseases were previously not reported in OA, but recent studies have indicated their high prevalence, especially in OA tribes living near cities1,3,15. Increased cardio-metabolic risks were also reported in aboriginal Torres Straits Islanders from Australia16, the Jaguapiru indigenous community in Brazil17 and the Rang Bothiya tribe in India18.\n\nMany OA tribes lead relatively more sedentary lifestyles compared to their hunter-gatherer ancestors. They can no longer and perhaps have no need to rely entirely on the forest and its resources for survival. Rapid development had given them easier access to high-calorie processed foods, which may have contributed to obesity and cardio-metabolic diseases.\n\nNational Health and Morbidity Survey 2015 reported obesity prevalence among the major ethnic groups in Malaysia to be 30.6%, which is comparable to the findings in this study (Temiar obesity = 32.4%); the national prevalence rate of diabetes was reported to be 22.9%19, which is very much higher than that of the Temiar (2.9%) in this study. Generally, reported prevalence rates of obesity among OA are still low. Nonetheless, the high prevalence rate of pre-diabetes reported among the Temiar (44.9%) indicates that rural OA communities are in dire need of awareness education and medical intervention.\n\nSaliva microbiome analysis revealed significant difference in microbial composition among men and women, where Prevotella was significantly higher in women. Hitherto, no studies have reported increased prevalence in women’s saliva microbiome. Prevotella in the gut has been associated with plant-rich diets and possibly with improved glucose tolerance20. Temiar women had a lower prevalence of pre-diabetes and insulin resistance, despite the majority of them presenting with either general or abdominal obesity. Perhaps the women consumed a traditional, indigenous diet, which is richer in plant-fibre and less meat compared to men. However, although Prevotella is a naturally occurring member of the oral microbiota21, it is also associated with inflammatory conditions such as rheumatoid arthritis and periodontal infections22.\n\nThe relative abundance of Bifidobacterium, healthy bacteria found in dairy products and used in probiotics, was shown to be elevated in Temiar men. While it is uncertain whether Temiar men were exposed to more dairy products than women, food taboos practiced among Temiar may contribute to the differences observed among gender23. Several studies that investigated oral microbiomes of urbanized cohorts in association with gender have reported no differences in oral microbiome profiles24–26. This may be explained by the relatively homogenous environment that urbanized cohorts were exposed to. Studies have shown that salivary microbiomes are most affected by environmental factors, as the oral microbiome of twins which were similar became highly dissimilar when they lived apart24.\n\nTemiar, on the other hand, lived in a traditional environment where men and women had different social standings. Men went out to the forest to hunt or forage while women stayed in the village with the children. They also observed certain food taboos, where the bush meat consumption of animals such as river terrapin, gibbons and porcupine were reserved only for men23.\n\nThis preliminary investigation suggested links between saliva microbiomes and gender where differences may be attributed to cultural, dietary and environmental factors. Even though the bacteria driving the differences in obesity and gender were of different species, it should be noted that most of the women were overweight/obese, which could be a confounding factor in gender-driven disparities in the saliva microbiome.\n\nStudies have suggested an association between obesity and altered oral microbiome27–29, concurring with the findings our study. Similar to our findings, a prior study found that Mogibacterium was reported to be significantly more abundant in an obese group30. Mycoplasma is a commensal, opportunistic oral pathogen reported to be elevated in overweight Temiar, perhaps due to dysbiosis of the oral microbiome. However, a significant difference was noted only when comparing overweight and underweight individuals. Both states are considered to be ‘unhealthy’ and thus assumed to be at dysbiosis.\n\nThe oral hygiene practices and oral health among Temiar were unknown, although due to their geographical isolation, it was highly unlikely they have regular access to dental health care. Our study revealed that the relative abundance of Proteobacteria, including Neisseria and Aggregatibacter, were decreased in smokers, compared to non-smokers. This was in line with the findings of Wu et al. (2016)31, although our study did not detect differences in the several other genera reported by them. This was probably due to the differences in sample size of both studies.\n\nWu et al. (2016) reported that Proteobacteria were associated with the breakdown of toxic hydrocarbons found in cigarette smoke, hence a depletion of the bacteria genus in smokers may prove detrimental to oral health31. An interesting difference noted was an increase in pathogenic anaerobe Streptococcus observed in their study31, whereas Hernandez et al. (2017)32 reported depletion of Streptococcus among betel-nut chewers, even after controlling for smoking. Our investigations revealed no significant difference, even though many of the Temiar smokers were also self-reported betel-nut chewers, a practice frequently associated with oral cancer33. Further investigations may be required to distinguish the effects of betel-nut chewing and smoking on the saliva microbiome.\n\n\nConclusion\n\nOur study revealed a high prevalence of cardio-metabolic diseases among the Temiar, including general and abdominal obesity, pre-diabetes and insulin resistance. Pre-hypertension was found highly prevalent across all age groups.\n\nInterestingly, the saliva microbiome profiles were significantly different for gender where the relative abundance of Prevotella, Capnocytophaga, Leptotrichia, Neisseria, Streptococcus and Bifidobacterium were concerned. Our study also noted a significant difference between the saliva microbiome compositions of underweight vs overweight and normal individuals. Mogibactericeae and Mycoplasma were found to be elevated in overweight individuals. The oral microbiome was not significantly different among non-diabetic, pre-diabetic and diabetic individuals. The microbiome profiles differed significantly among smokers and non-smokers where further investigation showed that Proteobacteria were significantly decreased in smokers. Investigation towards other health parameters such as pre-diabetes were inconclusive.\n\nThe sample size was limited, but findings from this pilot study warrants further and larger studies into other Malaysian indigenous tribes which may present unexpected findings perhaps attributed to differences in their cultural practices, lifestyle and diet.\n\n\nMethods\n\nThe study was approved by Ministry of Health Malaysia under National Medical Research Registry, MNDR ID #09—23-3913, Department of Orang Asli Development, Malaysia (JAKOA) and Monash University Human Research Ethics Committee (MUHREC). Before sampling, a courtesy visit to the Temiar elders in Kampong Pos Piah, Perak was conducted to explain the rationale of the study. Upon agreement to participate in our study, a medical team returned to the village on an agreed date and conducted health screening and sampling. Participants who were over 18 years old with no visible health ailments and able to provide informed consent were recruited for the study through convenience sampling, that is whoever who turned up and was eligible. Participants who were pregnant, with a history of alcohol/drug abuse, or with chronic illness (e.g. kidney failure, cancer, heart disease) were excluded from the study.\n\nThe consent form was read aloud by interviewers and queries were addressed before either a signature or thumbprint was provided as a sign of consent. A total of 72 Temiar provided informed consent to participate. Interviews were conducted in Bahasa Malaysia using a questionnaire12 to collect information about their socio-demography, medical history and diet. Height, weight, waist circumference and blood pressure were measured1. Participants were also examined by clinicians. Acanthosis negricans, which is darkening of the skin around the neck and creases of elbows indicative of insulin resistance, was noted.\n\nSaliva samples were collected in sterile 50ml polypropylene Falcon tube. Participants were requested to rinse their mouths with water thoroughly 30 minutes prior to collecting saliva. Venous blood samples were taken for biochemical analyses.\n\nBMI, waist circumference and blood pressure cut-off values were in accordance to WHO recommendations34. We measured their HbA1C and blood lipid levels (cholesterol, HDL, LDL, Triglyceride). We used TG/HDL ratio as a surrogate marker for insulin resistance with a cut-off value of 0.9-1.735.\n\nDNA was extracted from saliva using a modified high salt-method36. The V3-V4 region of the 16S rRNA gene were targeted, resulting in a PCR product of approximately 550 bp37.\n\nDNA sequencing was done by Genomics Facility in Monash University Malaysia on Illumina MiSeq to produce paired end reads of approximately 230 bp each.\n\nMicrobiome analysis was conducted on QIIME 1.938. Chimeras were filtered using UCHIME 1.39.339 before being aligned to Greengenes database V13.840. The reads were then clustered into operational taxonomic units (OTUs) with open-reference method at 97% similarity level using UCLUST41 in the QIIME pipeline. OTU clusters were assigned taxonomy with RDP classifier42. The reads were normalized and OTUs that were present at less than 0.05% were filtered off.\n\nAlpha diversity and beta diversity of the samples were reported using phylogenetic distance (PD) and UniFrac14, respectively. PCoA plots were generated to visualize beta diversity of the samples.\n\nStatistical analyses were completed on QIIME and R 3.4.4. Information taken from the mapping file included gender, BMI and smoking status. PERMANOVA, a non-parametric test was used to test for differences in median among the groups using weighted and unweighted UniFrac distance matrix Usi//ng the R packages vegan (v2.4-2), readr (v1.1.0) and dplyr (v0.5.0). Kruskal-Wallis test was used to test for differences in the relative abundance of OTUs among the different groups. A post-hoc test, Dunn’s test was done for pairwise comparison when testing factors like BMI and smoking, as they had more than two groups. False discovery rate, reported as q-value, was used to control for multiple hypothesis testing and was statistically significant at 5%.\n\n\nData availability\n\nSaliva microbiomes of the individuals in this study are available from the Sequence Read Archive, BioProject accession number PRJNA515166; https://identifiers.org/bioproject/PRJNA515166.\n\nAnthropometric data, along with the other variables measured, are available on figshare. DOI: https://doi.org/10.26180/5c453ff43588312.\n\nThe questionnaire used in this study is available on figshare. DOI: https://doi.org/10.26180/5c453ff43588312.\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 study was funded by Tropical Medicine and Biology Platform (TMB), Monash University Malaysia seed grant TMB -2016-HG3185140920-YLF/MP awarded to LFY and MEP. Ms Li-Fang Yeo is supported by a scholarship awarded by Jeffrey Cheah School of Medicine and Health Sciences, Monash 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\nAcknowledgements\n\nWe thank the Temiar community for their participation and the Department of Orang Asli Development (JAKOA) of Malaysia. We are especially grateful to Prof. Sunil K. Lal for helpful suggestions, Ms Yin Peng Lee for technical advice, and Dr Christina Yap for conducting biochemical analysis, Prof Daniel Reidpath for his assistance in statistical analysis on R, Dr Amreeta Dhanoa for editing the manuscript, Prof Yvonne Lim for advice and Dr Eustacia Lee and the University of Malaya team for participating in sample collection. Furthermore, our special thanks to Dr Badariah Ahmad and Dr Siti Noraida for participating in fieldwork.\n\n\nReferences\n\nPhipps ME, Chan KK, Naidu R, et al.: Cardio-metabolic health risks in indigenous populations of Southeast Asia and the influence of urbanization. BMC Public Health. 2015; 15: 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcColl H, Racimo F, Vinner L, et al.: The prehistoric peopling of Southeast Asia. Science. 2018; 361(6397): 88–92. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenjamin G: Temiar Religion, 1964-2012: Enchantment, Disenchantment and Re-enchantment in Malaysia’s Uplands. 2014. Reference Source\n\nStahringer SS, Clemente JC, Corley RP, et al.: Nurture trumps nature in a longitudinal survey of salivary bacterial communities in twins from early adolescence to early adulthood. Genome Res. 2012; 22(11): 2146–2152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen H, Jiang W: Application of high-throughput sequencing in understanding human oral microbiome related with health and disease. Front Microbiol. 2014; 5: 508. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi J, Quinque D, Horz HP, et al.: Comparative analysis of the human saliva microbiome from different climate zones: Alaska, Germany, and Africa. BMC Microbiol. 2014; 14(1): 316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakeshita T, Kageyama S, Furuta M, et al.: Bacterial diversity in saliva and oral health-related conditions: the Hisayama Study. Sci Rep. 2016; 6(1): 22164. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoodson JM, Hartman ML, Shi P, et al.: The salivary microbiome is altered in the presence of a high salivary glucose concentration. PLoS One. 2017; 12(3): e0170437. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanem WF, Scannapieco FA, Sabharwal A, et al.: Salivary inflammatory markers and microbiome in normoglycemic lean and obese children compared to obese children with type 2 diabetes. PLoS One. 2017; 12(3): e0172647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu Y, Chi X, Zhang Q, et al.: Characterization of the salivary microbiome in people with obesity. PeerJ. 2018; 6: e4458. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu J, Peters BA, Dominianni C, et al.: Cigarette smoking and the oral microbiome in a large study of American adults. ISME J. 2016; 10(10): 2435–2446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernandez BY, Zhu X, Goodman MT, et al.: Betel nut chewing, oral premalignant lesions, and the oral microbiome. PLoS One. 2017; 12(2): e0172196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuha N, Warnakulasuriya S, Vlaanderen J, et al.: Betel quid chewing and the risk of oral and oropharyngeal cancers: a meta-analysis with implications for cancer control. Int J Cancer. 2014; 135(6): 1433–43. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Use of glycated haemoglobin (HbA1c) in the diagnosis of diabetes mellitus. Abbreviated report of a WHO consultation 2011. Geneva: World Health Organization. 2013. Reference Source\n\nMostafa SA, Davies MJ, Morris DH, et al.: The association of the triglyceride-to-HDL cholesterol ratio with insulin resistance in White European and South Asian men and women. PLoS One. 2012; 7(12): e50931. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuinque D, Kittler R, Kayser M, et al.: Evaluation of saliva as a source of human DNA for population and association studies. Anal Biochem. 2006; 353(2): 272–277. PubMed Abstract \| Publisher Full Text\n\nPCR I: 16S Metagenomics Sequencing Library Preparation. Illumina. 2013.Reference Source\n\nCaporaso JG, Kuczynski J, Stombaugh J, et al.: QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010; 7(5): 335–336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdgar RC, Haas BJ, Clemente JC, et al.: UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011; 27(16): 2194–2200. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "44294", "date": "13 Mar 2019", "name": "Andres Gomez", "expertise": [ "Reviewer Expertise Microbial ecology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nResults The QIIME microbiome analyses should be briefly described as far as the bioinformatics pipeline used and its details: clustering of OTUS? OTU picking? amplicon sequence variants (DADA2-ASV)? These should be specifically detailed.\nIt does not seem that the rarefaction curve shows compete plateau, also what do the colors mean?\nPERMANOVA is used to mine for distinctions in microbiome composition.\nIt is unclear how kruskal-wallis test was used in conjunction with Unifrac - KW is used for pairwise comparisons in multivariate space, not for multivariate data.\nPERMANOVA results (P values) should be accompanied by R2 and, Pseudo F values also. PERMANOVA models used should also be reported.\n\"Kruskal-Wallis test revealed that the relative abundance of Prevotella, Capnocytophaga, Leptotrichia, Neisseria and Streptococcus\"\nWere these discriminant analyses made at genus or OTU/ASV levels? Also, it is recommended to show these results in box plots showing medians and variation of data, not in tables\n\nAuthors make several assertions as to the character and typically reported nature of the markers discovered, in the context of \"good/bad bacteria\" - but many of the claims made are in the context of gut microbiomes not oral communities. For instance: \"Prevotella, associated with consumption of plant-rich diets\" This claim may be true only for the gut microbiome not for oral communities. Also: \"The relative abundance of Bifidobacterium, the ‘good bacteria’ commonly found in dairy products, was found to be highly elevated in men\" - this may be associated with effect of dairy products in the gut not in the oral cavity.\n\n\"These commensal oral bacteria may become opportunistic pathogens in immune-compromised states.\" please cite\nFig 3- legends should indicate that a-b were based on Unw-UniFrac, and that C on W-UniFrac - Please depict % variation on axes\n\nIt is not clear how the post-hoc Dun's test were conducted-0 is this still based on PERMANOVA?\n\"The Kruskal-Wallis test generated by QIIME revealed no significant difference.\" This is not clear - compared to what?\n\n\"There was a significant difference between waist circumference and saliva microbiome composition (weighted UniFrac, p-value = 0.022). \" This is not clear - you mean differences between the microbiomes of subjects with different waist circumference?\nQIIME is just a software with plugins to perform statistical analyses - it is not clear when authors are using qiime or R and how these platforms would yield different results. Please avoid jargon as \"generated by QIIME\"\n\"The saliva microbiome showed no significant difference between former smokers and never-smokers, suggesting perhaps the saliva microbiome may recover partially to an environment prior to smoking\". This is unclear but also speculative.\n\nThe discussion is highly speculative and should be toned down, mainly as far as the hypotheses behind the differences reported.\nAuthors also seem to interchangeably extrapolate hypothesis on oral microbiome changes with gut microbiome changes in the context of lifestyle and diet (e.g. Prevotella) although diet may also be linked to oral microbiome modulation, this relationship is less clear, and taxa such as Prevotella, abundant in both gut and oral cavity, cannot be assumed to be modulated by diet in both sites\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4489", "date": "09 May 2019", "name": "Li-Fang Yeo", "role": "Author Response", "response": "Details on the bioinformatics pipeline will be addressed in version 2. The different colours on the rarefaction curve represent different individuals. Other metrics such as Shannon and Chao1 shows an obvious plateau. We felt that PD was more informative because it included phylogenetic distances. Our statistics were done mostly with reference to this paper: Wu, J., Peters, B.A., Dominianni, C., Zhang, Y., Pei, Z., Yang, L., Ma, Y., Purdue, M.P., Jacobs, E.J., Gapstur, S.M. and Li, H., 2016. “Cigarette smoking and the oral microbiome in a large study of American adults”. The ISME journal. https://www.nature.com/articles/ismej201637 However, more recent methods such as using balances and log ratio shall be considered in a larger, extended study. Test scores for PERMANOVA results shall be addressed in version 2 as will several questions that were unclear in version 1. To date, we have none found literature that was comparable (ie. Having Bifidobacterium elevated in men, Prevotella elevated in women). Hence we attempted to suggest possible explanations to this observation. The discussion will be re-worded to take into account reviewer’s comments." }, { "c_id": "4656", "date": "20 May 2019", "name": "Andres Gomez", "role": "Reviewer Response", "response": "Dear authors,Could you please provide point by point responses to the comments raised in the previous version, along with where changes were made in the manuscript?That would facilitate reviewing of the new manuscript." }, { "c_id": "4657", "date": "20 May 2019", "name": "Li-Fang Yeo", "role": "Author Response", "response": "Dear reviewer, As per request: 1. The QIIME microbiome analyses should be briefly described as far as the bioinformatics pipeline used and its details: clustering of OTUS? OTU picking? amplicon sequence variants (DADA2-ASV)? These should be specifically detailed. Microbiome analysis pipeline edited and detailed in Methods section. 2. It does not seem that the rarefaction curve shows compete plateau, also what do the colors mean? Rarefaction curve now shows Shannon index instead of phylogenetic diversity for better indicator of plateau. Each colour is a biological sample. 3. It is unclear how kruskal-wallis test was used in conjunction with Unifrac - KW is used for pairwise comparisons in multivariate space, not for multivariate data. KW test was conducted on an OTU table, not UniFrac. Refer to Ying & Sun (2017) https://www.sciencedirect.com/science/article/pii/S2352304217300351 Edits made in Results section. 4. PERMANOVA results (P values) should be accompanied by R2 and, Pseudo F values also. PERMANOVA models used should also be reported. Pseudo F values are reported. R2 value not available for PERMANOVA but available for adonis. All available values are reported in supplementary material. 5. \"Kruskal-Wallis test revealed that the relative abundance of Prevotella, Capnocytophaga, Leptotrichia, Neisseria and Streptococcus\" Were these discriminant analyses made at genus or OTU/ASV levels? Also, it is recommended to show these results in box plots showing medians and variation of data, not in tables These analyses were made at OTU level 6, equivalent to genus level. Addressed in Results section. Also, due to time constraint and admittedly technical limitations on our part, the results will be shown in tables for this paper. Reviewer's advice are definitely well-received and will be considered for our next paper. 6. Authors make several assertions as to the character and typically reported nature of the markers discovered, in the context of \"good/bad bacteria\" - but many of the claims made are in the context of gut microbiomes not oral communities. We have acknowledged the reviewer's comments and rephrased the discussion to be more cautious and less speculative. 7. \"These commensal oral bacteria may become opportunistic pathogens in immune-compromised states.\" please cite Idris A, Hasnain SZ, Huat LZ, Koh D. Human diseases, immunity and the oral microbiota—Insights gained from metagenomic studies. Oral Science International. 2017 Jul;14(2):27–32. Cited in-text as well. 8. Fig 3- legends should indicate that a-b were based on Unw-UniFrac, and that C on W-UniFrac - Please depict % variation on axes Done. 9. It is not clear how the post-hoc Dun's test were conducted-0 is this still based on PERMANOVA? Post-hoc Dunn's test was conducted using an OTU table. Permanova does not conduct pairwise comparison hence Dunn's test was chosen to test for differences between people with different BMI status (underweight, normal, overweight). And the test revealed that underweight individuals had microbiomes that was significantly different from both normal and overweight individuals. 10. \"The Kruskal-Wallis test generated by QIIME revealed no significant difference.\" This is not clear - compared to what? 12. QIIME is just a software with plugins to perform statistical analyses - it is not clear when authors are using qiime or R and how these platforms would yield different results. Please avoid jargon as \"generated by QIIME\" For 10 & 12, previously qiime and R yielded different results (one not significant, one significantly different) for oral microbiomes and BMI. Upon closer inspection we realized this happened because of mismatching samples and OTU table upon input to R. We checked the other sets of data and the qiime results matches with R. Changes addressed in Results section. 11. \"There was a significant difference between waist circumference and saliva microbiome composition (weighted UniFrac, p-value = 0.022). \" This is not clear - you mean differences between the microbiomes of subjects with different waist circumference? The above sentence has been rephrased to \"There was a significant difference in the saliva microbiome of Temiar who had a healthy waist circumference compared to those with abdominal obesity.\" 13. \"The saliva microbiome showed no significant difference between former smokers and never-smokers, suggesting perhaps the saliva microbiome may recover partially to an environment prior to smoking\". This is unclear but also speculative. The discussion is highly speculative and should be toned down, mainly as far as the hypotheses behind the differences reported. Authors also seem to interchangeably extrapolate hypothesis on oral microbiome changes with gut microbiome changes in the context of lifestyle and diet (e.g. Prevotella) although diet may also be linked to oral microbiome modulation, this relationship is less clear, and taxa such as Prevotella, abundant in both gut and oral cavity, cannot be assumed to be modulated by diet in both sites This comment has been well-received and edits have been done to Discussion section. TQ." } ] }, { "id": "46035", "date": "10 Apr 2019", "name": "Siti Nursheena Mohd Zain", "expertise": [ "Reviewer Expertise Parasitology and molecular parasitology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting work looking at non-communicable disease risks link to microbiome in saliva as an indicator. The discussion could be written better to pull the results together. What is missing is a comparative study with another sedentary population. I would like if the conclusion is added with recommendations to improve the issue facing the Temiar tribe. Noted that sample size is small to make small but still warrants some finding to improve their health status.\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/8-175
https://f1000research.com/articles/8-742/v1	28 May 19	{ "type": "Research Article", "title": "Safety and efficacy of percutaneous nephrolithotomy in patients with a single functioning kidney compared to patients with bilateral functioning kidneys", "authors": [ "Riyadh R. Al-Toma" ], "abstract": "Background: Renal stones for patients with a single functioning kidney are considered a great challenge for urologists. Different treatment modalities are used for those with a single functioning kidney, including shock wave lithotripsy, retrograde intra-renal surgery and percutaneous nephrolithotomy (PCNL).\n\nThis study aimed to compare the effectiveness and safety outcomes of PCNL in patients with a single kidney compared to those with bilateral kidneys. Methods: A prospective comparative study conducted in Urology department of Safeer Al-Imam Al-Hussein hospital in Karbala city-Iraq through the period from 1st of March, 2015 to 30th of September, 2018 on sample of 173 patients with renal stones surgically operated with PCNL categorized into two groups; group I included 51 patients with a single functioning kidney and group II included 122 patients with bilateral functioning kidneys. Results: The mean age of group I patients was 42.76 years which was significantly higher than mean age of group II patients of 36.54 years (p=0.01). No significant differences were observed between group I and group II patients regarding gender, pre and intraoperative characteristics. Postoperative stone clearance, bleeding, renal function, organ injury and sepsis were not significantly different between two.\n\nConclusions: PCNL is effective and safe surgical procedure for treatment of renal stones of patients with a single functioning kidney.", "keywords": [ "Renal stone", "Single functioning kidney", "Bilateral functioning kidneys", "Percutaneous nephrolithotomy", "Karbala" ], "content": "Introduction\n\nUrolithiasis is a frequent disorder affecting the urinary system. Globally, urinary stones are represented at a prevalence of 5–12% in males and 4–7% in females1. Percutaneous nephrolithotomy (PCNL) is the best choice for treatment of stones, giving a high stone-free rate and increased safety when compared to other techniques2. The PCNL is used for management of stones sized 2 cm3 and above3. In spite of these PCNL advantages, it is often accompanied by many complications, such as bleeding, collecting system injury, urinary leakage, infection, kidney damage and death4. Solitary kidney is defined as the condition where an individual has a single functioning kidney as compared to normally two kidneys. The incidence of stones in patients with solitary and bilateral kidneys are same rate5. The complications reported during and after PCNL of patients with solitary kidney were uncontrolled bleeding, need for angio-embolization or nephrectomy and subsequent need for kidney transplant6.\n\nThis study aimed to compare the effectiveness and safety outcomes of PCNL in patients with single functioning kidney in comparison to PCNL outcomes in patients with bilateral functioning kidneys.\n\n\nMethods\n\nThis study was a prospective comparative study conducted in the Urology Department of Safeer Al-Imam Al-Hussein hospital in Karbala city, Iraq, through the period from 1st of March, 2015 to 30th of September, 2018. We recruited patients with and without nephrostomy tubes in this study.\n\nInclusion criteria included renal stone more than 2 cm in size, negative culture of urine and patients with single functioning. Exclusion criteria included patients with full staghorn calculi, patients with single functioning kidney with deteriorated health, concomitant angiomyolipoma, coagulopathy diseases, collecting system perforation, severe intraoperative bleeding, elevated creatinine level and ectopic or fused kidney.\n\nParticipants were selected if they fit the aforementioned eligibility criteria and if they attended hospital. No efforts were made to control bias in recruitment or analysis. This study included a convenience sample of 173 patients (recruited face to face in the clinic and also via telephone and social media), with renal stones who underwent PCNL. Patients were arranged into two groups: group I included 51 patients with single functioning kidney and group II included 122 patients with bilateral functioning kidneys.\n\n\nData sources and collections\n\nAssessment of patients was done by the researcher during preoperative, operative and postoperative periods. Full history and examination of patients was firstly done by the researcher and then patients were sent to the Laboratory and Radiology departments of hospital to undergo complete blood and radiological investigations. Diagnosis of renal stones was conducted by the researcher depending on clinical features, investigations and imaging techniques.\n\n\nProcedures\n\nPre- and postoperative intravenous urography, and preoperative ultrasound and plain x-ray of the kidney were done preoperatively for all patients. PCNL surgery was initiated after giving patients general anesthesia in the lithotomy position and inserting a 6 F open-end ureteral catheter via cystoscopy. PCNL was conducted by a urologist and included percutaneous puncture of the pelvicalyceal system, arrangement of the tract and the fragmentation or removal of stones. At the end of the procedure, stone clearance was confirmed by endoscopy and fluoroscopy and the ureteric catheter was removed. In tubed PCNL patients, a nephrostomy tube was positioned through the Amplatz sheath and fixed to the skin and the nephrostomy was clamped for 12 hours. In tubeless PCNL, after removal of the Amplatz sheath the wound was compressed for two minutes and then sutured with one-stitch non-absorbable suture followed by dressing without insertion of a nephrostomy tube.\n\n\nStatistical methods\n\nStatistical analysis was implemented using SPSS version 20. For analysis of categorical variables, chi-square and Fisher's exact tests were applied; for continuous variables, the independent sample t-test was used. P=0.05 was considered to indicate statistical significance.\n\n\nEthical considerations\n\nEthical considerations were included a written informed consent from each patient before enrolling in the study and before PCNL surgery; approval was taken from the authorities of the Safeer Al-Imam Al-Hussein hospital (code:77331).\n\n\nResults\n\nThe Mean age of group I patients was 42.76 years, which was significantly higher than mean age of group II patients of 36.54 years (p=0.01). Male gender patients in two study groups was predominant (52.9% vs. 53.3%); however, there was no significant difference between two study groups regarding gender (p=0.96) (Table 1). Postoperatively, the stone-free and residual-stone rates of group I patients were 90.2% and 9.8%, respectively, while for group II patients, they were 94.3% and 5.7%, respectively, with no significant difference between two study groups regarding the stone clearance (p=0.34). Regarding postoperative PCNL complications, no significant differences were observed between group I and group II patients in relation to postoperative bleeding (p=0.79) impaired renal function (p=0.84), organ injury (p=0.36) and sepsis (p=0.64) (Table 2). Underlying data for this study are available from Zenodo7.\n\n\nDiscussion\n\nThe current study showed a significant difference in mean age between patients with solitary kidneys and those with two kidneys. The patients with single kidneys were older than those with bilateral kidneys. This finding is consistent with the results of Basiri et al.8 in Iran, which reported mean age of 42.1 years for single kidney in comparison to 38.5 years for double kidneys. No significant differences were observed between our study groups regarding gender, although the male patients were more than female patients. These findings are similar to previous Iraqi studies9,10.\n\nThe preoperative and intraoperative characteristics of both study groups patients were not significantly different. These findings agree with results of many prior studies like those of Yaycioglu et al.11 in Turkey and Agrawal et al.12 in India. PCNL intraoperative difficulties might be observed among patients with solitary kidney like some problems in PCNL approach and tracts13.\n\nAnalysis of postoperative outcomes showed no significant difference in clearance rate between patients with single and bilateral kidneys (p=0.34). Similarly, Haberal et al.14 revealed that the postoperative stone free rate was similar for both solitary and bilateral kidneys. The postoperative complications were not significantly different between both study groups. This finding coincides with the results of Wong et al.15 in the UK and Akman et al.16 in Turkey, which documented that PCNL is a safe procedure for patients with solitary kidney with acceptable complication rates.\n\nTo conclude, PCNL is effective and safe for treatment of renal stones of patients with single functioning kidney.\n\n\nData availability\n\nZenodo: Safety and efficacy of percutaneous nephrolithotomy. https://doi.org/10.5281/zenodo.28727787.\n\nThis project contains raw data for each patient assessed in this study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHuang WY, Chen YF, Carter S, et al.: Epidemiology of upper urinary tract stone disease in a Taiwanese population: a nationwide, population based study. J Urol. 2013; 189(6): 2158–2163. PubMed Abstract \| Publisher Full Text\n\nAntonelli JA, Pearle MS: Advances in percutaneous nephrolithotomy. Urol Clin North Am. 2013; 40(1): 99–113. PubMed Abstract \| Publisher Full Text\n\nMehmet NM, Ender O: Effect of urinary stone disease and its treatment on renal function. World J Nephrol. 2015; 4(2): 271–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichel MS, Trojan L, Rassweiler JJ: Complications in percutaneous nephrolithotomy. Eur Urol. 2007; 51(4): 899–906; discussion 906. PubMed Abstract \| Publisher Full Text\n\nDamera R, Karthik K: Percutaneous Nephrolithotomy in Patients with Solitary Kidney– Svims Experience. IOSR Journal of Dental and Medical Sciences. 2016; 15(11): 100–103. Reference Source\n\nBucuras V, Gopalakrishnam G, Wolf JS Jr, et al.: The Clinical Research Office of the Endourological Society Percutaneous Nephrolithotomy Global Study: nephrolithotomy in 189 patients with solitary kidneys. J Endourol. 2012; 26(4): 336–341. PubMed Abstract \| Publisher Full Text\n\nAaltoma R: Safety and efficacy of percutaneous nephrolithotomy in patients with sfk compared to patients with bfk. 2019. http://www.doi.org/10.5281/zenodo.2872779\n\nBasiri A, Shabaninia S, Mir A, et al.: The safety and efficacy of percutaneous nephrolithotomy for management of large renal stones in single- versus double-functioning kidney patients. J Endourol. 2012; 26(3): 235–238. PubMed Abstract \| Publisher Full Text\n\nAl-Aridy HM: Percutaneous nephrolithotomy for renal calculi: A single surgeon experience. The Iraqi Postgraduate Medical Journal. 2013; 12(4): 573–580. Reference Source\n\nAbid AF: Feasibility and Efficacy of Percutaneous Nephrolithotomy, Initial Experience. The Iraqi Postgraduate Medical Journal. 2016; 15(3): 334–337. Reference Source\n\nYaycioglu O, Egilmez T, Gul U, et al.: Percutaneous nephrolithotomy in patients with normal versus impaired renal function. Urol Res. 2007; 35(2): 101–105. PubMed Abstract \| Publisher Full Text\n\nAgrawal MS, Aron M, Asopa HS: Endourological renal salvage in patients with calculus nephropathy and advanced uraemia. BJU Int. 1999; 84(3): 252–256. PubMed Abstract \| Publisher Full Text\n\nVicentini FC, Gomes CM, Danilovic A, et al.: Percutaneous nephrolithotomy: Current concepts. Indian J Urol. 2009; 25(1): 4–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaberal HB, Çıtamak B, Bozacı AC, et al.: Percutaneous Nephrolithotomy in Solitary Kidneys: 17 Years of Experience. Urology. 2017; 109: 55–59. PubMed Abstract \| Publisher Full Text\n\nWong KA, Sahai A, Patel A, et al.: Is percutaneous nephrolithotomy in solitary kidneys safe? Urology. 2013; 82(5): 1013–1016. PubMed Abstract \| Publisher Full Text\n\nAkman T, Binbay M, Tekinarslan E, et al.: Outcomes of percutaneous nephrolithotomy in patients with solitary kidneys: a single-center experience. Urology. 2011; 78(2): 272–276. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62595", "date": "21 May 2020", "name": "Panagiotis Kallidonis", "expertise": [ "Reviewer Expertise Endourology - Urolithiaiss" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 is presenting a study comparing the outcome of PCNL in patients with single functioning kidneys to patients with both kidneys.\n\nIntroduction: “The PCNL is used for management of stones sized 2 cm3 and above.” Please consider rephrasing. In the EAU guidelines, PCNL is the gold standard for stones >2cm (maximal diameter). Stone volume is more appropriate for reporting but most of the literature considers the maximal diameter.\nMethods: What about institutional board approval for the conducting the study? Informed consent from the patients? “We recruited patients with and without nephrostomy tubes in this study.” Should added to the inclusion-exclusion criteria. Were the cases consecutive?\n\n“…patients with single functioning kidney with deteriorated health.” Please explain.\n\nIt is not clear which imaging studies were considered for the evaluation of the patients. CT scans are more appropriate for accurate depiction of the stone size/location.\n\nWhich was the approach for evaluating the patients with single functioning but with the presence of non-functional contralateral kidney?\n\nPlease provide a more detailed description of the technique. Was it the same for all cases? It is not clear which was the position of the patient during the PCNL (percutaneous access). What was the size of the tract? What kind of lithotripter was used? What was the size of the nephrostomy? Which were the criteria for tubeless or non-tubeless technique? I do not really understand why to clamp the nephrostomy tube for the first 12 hours. The nephrostomy tube is placed to ensure drainage not to tamponade the tract.\n\nWhat was the follow-up schedule for these patients? Which imaging techniques were used for evaluating the stone-free status and when? Definition of stone-free status? Clavien-classification of the complications?\n\nResults: A lot of information is missing. Stone location and the presence of multiple stones are important. Multitract PCNL may have been necessary, Operative time? Hospitalization time? The current information is very limited for the reader to follow the results.\n\nDiscussion: There are several articles on PCNL and solitary kidneys that could be considered for discussion.\n\nBased on the experience of the author, which are important parameters for safety and efficacy of PCNL in cases with solitary kidneys? Technical points to propose?\n\nWhich are the string points of the study?\n\nWhat the study adds to the available literature?\n\nLimitations of the study?\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "64736", "date": "29 Jun 2020", "name": "Biagio Barone", "expertise": [ "Reviewer Expertise Endourology - Urolithiasis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presents a study comparing effectiveness and safety of PCNL in single functioning kidney versus both.\nThe study, despite does not be particularly original, could further enrich the literature on this issue. Introduction:\n“Percutaneous nephrolithotomy (PCNL) is the best choice for treatment of stones […]” is too affirmative and absolute. Multiple articles report the use of RIRS for even larger stones size with satisfying results. It would be better to add “for stones > 2cm” for example.\n\n“The PCNL is used for management of stones sized 2 cm3 and above”: refer to guidelines\nMethods:\n“We recruited patients with and without nephrostomy tubes in this study”. Move to inclusion criteria\n\n“patients with single functioning”. Define single functioning kidney (previous evaluation, solitary kidney…). How did you evaluate those kidneys? Renal scintigraphy?\n\n“if they attended hospital”: This is unnecessary as you have already defined the population recruited for the study as patients of your hospital.\n\nCould you add further information on the surgery? (tract size, type of lithotripter, same or different surgeon). Did you use a standardized technique?\n\nWhich were the indications for tubeless PCNL?\n\nWhich was the mean duration of postoperative follow up?\nResults:\nI would define the gender prevalence as slightly predominant considering the thin difference in percentages\n\nHow did you define the residual stone?\n\nAn important addition could be operative and hospitalization time, if data are available\n\nDiscussion:\nEnrich the discussion with more literature on the issue. Report limitations of the study and do not rush the conclusion\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/8-742
https://f1000research.com/articles/8-233/v1	28 Feb 19	{ "type": "Software Tool Article", "title": "Assessing drug target suitability using TargetMine", "authors": [ "Yi-An Chen", "Erika Yogo", "Naoko Kurihara", "Tomoshige Ohno", "Chihiro Higuchi", "Masatomo Rokushima", "Kenji Mizuguchi", "Erika Yogo", "Naoko Kurihara", "Tomoshige Ohno", "Chihiro Higuchi", "Masatomo Rokushima" ], "abstract": "In selecting drug target candidates for pharmaceutical research, the linkage to disease and the tractability of the target are two important factors that can ultimately determine the drug efficacy. Several existing resources can provide gene-disease associations, but determining whether such a list of genes are attractive drug targets often requires further information gathering and analysis. In addition, few resources provide the information required to evaluate the tractability of a target. To address these issues, we have updated TargetMine, a data warehouse for assisting target prioritization, by integrating new data sources for gene-disease associations and enhancing functionalities for target assessment. As a data mining platform that integrates a variety of data sources, including protein structures and chemical compounds, TargetMine now offers a powerful and flexible interface for constructing queries to check genetic evidence, tractability and other relevant features for the candidate genes. We demonstrate these features by using several specific examples.", "keywords": [ "disease", "drug assessment", "genetic variation", "tractability" ], "content": "Introduction\n\nA drug discovery project typically begins with the identification of a target molecule. In evaluating potential drug targets, several factors must be taken into account: linkage to disease, tractability (the possibility of finding small molecule compounds with high affinity), potential side effects, novelty, as well as the competitiveness in the market (Figure 1). Among these factors, the linkage to disease and the tractability are particularly important in terms of the drug efficacy, and become key factors in whether or not the pharmaceutical research and development (R&D) is successful when selecting drug targets1,2. The most important part of the linkage to disease is genetic associations for the disease or relevant traits. According to analyses reported by AstraZeneca and GlaxoSmithKline, the success rate of such R&D is increased when the choice of the selected target is supported by genetic evidence. The report from AstraZeneca shows that 73% of projects with some genetic linkage of the target to the disease indication in Phase II were active or successful compared to 43% of projects without such data3, while the analysis results from GlaxoSmithKline suggest that selecting genetically supported targets could double the success rate in clinical development4. Several existing resources provide information about genetic evidences, such as DisGeNET5, Open Targets6, and Pharos7. However, a simple list of genes with genetic linkage to the disease is often insufficient for evaluating the disease rationale fully, and additional information and analysis such as pathway enrichment analysis will be needed to assess other aspects of target suitability (e.g. drug mechanisms and safety). In addition, few resources provide tractability information, with the recent update of Open Targets being an exception.\n\nLinkage to disease, tractability and adverse event risk are among the major factors to assess the suitability of novel target candidates. Much of the evidences regarding these factors are available in public domain resources.\n\nTo address these issues, we have updated TargetMine8, a data warehouse for assisting target prioritization, and improved its functionalities for target assessment, particularly in small molecule drug discovery. TargetMine8 utilizes the InterMine framework9 and facilitates flexible query construction spanning a wide range of integrated data sources including those relevant for evaluating linkage to disease and tractability. More specifically, we have integrated new data sources for genetic disease associations including ClinVar, dbSNP, and 1000 Genome Project, incorporated more details of the genome wide association studies from the GWAS catalog, and improved the data model overall to enable more efficient data mining. The new version provides a user-friendly and yet powerful interface to explore the disease rationale for human genes and helps prioritize the candidate genes in terms of both the genetic evidence and target tractability.\n\n\nMethods\n\nTargetMine8 is based on the InterMine framework, an open-source data warehouse system designed for biological data integration9. In this update, we added a few customized data sources by defining new data models and implementing new data parsers. Details of how we designed the data models are described in the following sub-sections.\n\nThe GWAS catalog, founded by NHGRI, is a curated archive of the published genome wide association studies10. We had tried to associate genes to related diseases using the GWAS catalog in the former release of TargetMine11. To annotate disease terms to a trait or study, we first chose the disease ontology (DO)12,13 and then manually assigned the terms with the assistance of some text matching approaches. However, this process required some knowledge and involved a lot of manual examinations. Thus, it became difficult to keep updating regularly. Fortunately, the curation team started to use experiment factor ontology (EFO)14 to describe the curated GWAS traits in the recent implementation15. EFO covers several domain-specific ontologies that facilitate easier data integration. In our new implemented model, we replace DO terms with EFO terms and also incorporate some more information from each study (Figure 2). SNP annotations and details of EFO terms are retrieved from the dbSNP database and EFO, respectively.\n\nThe colored lines indicate how the genes and diseases/phenotypes are associated in the post processing step.\n\nClinVar is a public archive of the relation between human variations and phenotypes16,17. As defined by ClinVar, a “Variation” could be a single variant, a compound heterozygote, or a complex haplotype. If a haplotype consists of multiple alleles, each allele is assigned with an independent identifier. On the other hand, the same allele could be the member of a different haplotype, thus the relation between the “Variation” and “Allele” is a many-to-many association. An “Allele” is supposed to describe a specific change of a variation, e.g. G>A. However, the SNP entries in dbSNP sometimes merge different combinations of variations (alleles) together if the variations occur at the same genomic position. Thus, an “SNP” entity may contain multiple “Allele” entries in the data model (Figure 2). Here, we only retrieve the SNP identifier, and the rest of the annotations are integrated from the dbSNP database. The structural variations which reference the dbVar records are not included in the current version. In addition, those alleles which were not assigned with any dbSNP or dbVar identifiers were treated as SNP entities and were stored in TargetMine8 using the information provided by ClinVar. Most of the data were processed from tab delimited files, while some information that were not available in the tab delimited files were processed from XML files. MedGen terms, which are used to integrate the human medical genetic information at NCBI (https://www.ncbi.nlm.nih.gov/medgen/), were adopted to describe diseases and phenotypes.\n\ndbSNP is a database which archives short human genetic variations. We first performed a whole data dump to a relational database, and then made queries to extract the necessary information into a flat table. These data include genomic position (based on genome assembly GRCh38), reference mRNA, nucleotide variation, reference protein, and amino acid variation, if available. SNP to gene is a many-to-many relationship, thus we introduce an intermediate class named “VariationAnnotation” to associate them together (Figure 2). Although the InterMine framework is capable of incorporating whole SNP entries in dbSNP, the integration takes a few days to finish. Considering the frequency that we update TargetMine8 (once a month), it is not very practical to spend a few days doing the integration. As a tradeoff, we decided to store only a subset of SNPs. Only those SNPs which are related with GWAS associations or clinical assertions, or those where there is an associated publication, are selected for storage in TargetMine8.\n\nPopulation specific genetic variation frequency is important for evaluating drug efficacy. We preprocessed the frequency data from several data sources, including the Human Genetic Variation Database (HGVD)18, the integrative Japanese Genome Variation Database (1KJPN)19 (download from the archive in National Bioscience Database Center), the Exome Variant Server (EVS)20, and the 1000 Genomes Project (1KGP)21,22. At the moment, we only incorporate the population specific frequency for those SNPs stored in TargetMine8.\n\nOur implementation allows us to associate the genetic phenotype (disease) and the gene via the GWAS or ClinVar dataset, or moreover the relation that is implied from the disease related MeSH (Medical Subject Headings, https://www.ncbi.nlm.nih.gov/mesh) terms assigned to the correlated publications of the SNPs. In order to make a shortcut and to summarize the available information, we perform post-processing and store the results using a new class named “GeneDiseasePair”. At the moment, there are three types of shortcuts. Gene to SNP to GWAS to EFO terms for GWAS catalog data (the red lines in Figure 2). Gene to SNP to clinical assertions to disease (MedGen) terms (the green lines in Figure 2). And Gene to SNP to publication to MeSH terms (the blue lines in Figure 2). The “GeneDiseasePair” class also includes correlated information including ontology terms, studies, SNPs and publications. These improvements in the data model facilitate quick access from a gene to the associated diseases, annotated by different data sources.\n\nTargetMine8 is a Java-based web application that runs on Apache Tomcat. The user interface communicates with the integrated data stored in PostgreSQL, a relational database.\n\n\nUse cases\n\nTo demonstrate the effectiveness of the new version of TargetMine8 in evaluating linkage to disease, we conducted a feasibility study, taking human PCSK9, proprotein convertase subtilisin/kexin type 9, as a typical case. The PCSK9 gene encodes a protein that promotes degradation of low-density lipoprotein (LDL) receptors in hepatocytes, thereby elevating or maintaining LDL cholesterol levels in the blood. Mutations in this gene are shown to be associated with familial hypercholesterolemia23, and monoclonal antibodies to PCSK9 have been launched on the market as drugs for hypercholesterolemia with and without genetic predispositions24,25.\n\nFigure 3A demonstrates a schematic representation of the searching protocol for genetic disease associations with TargetMine8. We first went to a gene report page by searching for the PCSK9 gene from the top page of TargetMine8 (not shown). From the gene report page, we got information of genetic disease associations (Figure 3B) as well as many other basic or advanced characteristics such as orthologous genes and upstream transcription factors. The results table of genetic disease associations for PCSK9 enabled us to confirm that a number of SNPs relevant to this gene have been reported to be associated with plasma LDL cholesterol levels, hypercholesterolemia, or coronary artery disease. By clicking the record of association between “low density lipoprotein cholesterol measurement” and PCSK9 in the GWAS catalog section (Figure 3B), we moved to a “gene disease pair” page and checked the details of the GWAS record, including the information on samples, statistical significance and publications (Figure 3C). Clicking on the SNP identifier (e.g., rs2479409) redirected us to an SNP report page containing the individual SNP basic information (allele, function, literature) and allele frequencies of different human populations (from 1000 Genome Project26 and others, not shown in the figure). Similarly, we examined the associations between “Hypercholesterolemia, autosomal dominant, 3” and PCSK9 from the ClinVar section in the table (Figure 3B) and got the details of the ClinVar record such as clinical assertions and publications (Figure 3D). The publications here reported mutations in PCSK9 as a cause of autosomal dominant hypercholesterolemia23 (not shown), as mentioned above.\n\n(A) Outline of the procedure for searching. (B) A screenshot of the summary of Genetic disease associations of PCSK9. (C) GWAS records of a pair of PCSK9 and low density lipoprotein cholesterol measurement. (D) ClinVar records of a pair of PCSK9 and hypercholesterolemia, autosomal dominant, 3.\n\nWe performed another feasibility study to examine whether TargetMine8 provides informative evidence to assess target tractability for small molecules. In this case we also used PCSK9 as an example because no potent small molecule inhibitors for this protein have been reported so far in spite of the intensive research activities of many laboratories27, indicating that PCSK9 is not a highly tractable target.\n\nFigure 4A shows a schematic diagram of the procedure of querying tractability with TargetMine8. We first went to the protein report page of PCSK9 and found the bioactive compounds targeting this protein. As we expected, it was revealed that no potent compounds could be found in the ChEMBL database, and the lowest IC50 value was 440 nM (CHEMBL3923422) (Figure 4B). On the PCSK9 protein report page, we also checked the experimentally determined 3D structures, referred to as “protein structure regions” in TargetMine8, and identified several Protein Data Bank (PDB) entries for this protein (Figure 4C). Then, we moved to the “Protein Structure” page of a specified PDB ID (2p4e in this case) and found that in the “DrugEBIlity” table (from the DrugEBIlity database), some domains of the PCSK9 protein had positive Ensembl scores (Figure 4D), which are not ligand-based, but structure-based tractability scores. This result indicates that PCSK9 protein may contain some sites/pockets that can bind small molecules, although Ensemble scores of DrugEBIlity may need to be further validated.\n\n(A) Outline of the procedure for searching. (B) Protein structure regions and their Ensembl scores calculated by DrugEBIlity. (C) Compounds with bioactivity for PCSK9.\n\nCollectively, we were able to confirm that the new version of TargetMine8 can quickly provide lines of evidences to assess linkage to disease and target tractability of PCSK9, and that the gathered data correctly reflected the real world situation; namely, it has been a challenge to obtain potent small molecule inhibitors for PCSK9, whereas antibody drugs for this protein have been successfully developed and marketed recently.\n\nTo assess the utility of the new update of TargetMine8 for prioritizing candidate targets, we conducted a case study where we employed a list of genes associated with hypercholesterolemia in literature. We tentatively defined three key properties of a novel drug target suitable for small molecules as follows: (1) being associated with hypercholesterolemia via SNPs (GWAS catalog, ClinVar, or dbSNP-Pubmed; see Materials and Methods), (2) having greater than or equal to 50% of protein 3D structures with positive Ensemble scores (DrugEBIlity), and (3) having no reported (ChEMBL) potent small molecule inhibitors (IC50 or EC50 ≤ 100 nM).\n\nWe first searched PubMed using the term “hypercholesterolemia” (from 2017/1/1 to 2018/9/10) and curated the resultant literature with the “Pubtator” text-mining tool28, resulting in 510 human genes (Figure 5A). We then selected the genes meeting the requirements defined above using the “Query Builder” in TargetMine8. Figure 5B shows an overview of the actual query, which aimed to extract the genes with gene evidences obtained from the GWAS catalog, where “Mapped Trait” contained “LDL cholesterol”, “total cholesterol”, or “low density lipoprotein cholesterol”, from ClinVar where “Reported phenotype Info” contains the term “Hypercholesterolemia”, and from dbSNP where “Mesh Terms Name” of related articles contains “Hypercholesterolemia”. Thus, the new implementation enabled us to filter objects on complex conditions with a user-friendly, intuitive graphical interface.\n\n(A) Gathering hypercholesterolemia-related genes from article information in PubMed. (B) Prioritizing hypercholesterolemia-related genes with TargetMine to identify novel targets for small molecule drugs. Top prioritized genes were defined as those that met all of the following three requirements: 1) more than or equal to 50% of protein 3D structures (PDB IDs) having positive Ensembl scores, 2) no potent bioactive compounds (EC50 or IC50 ≤ 100 nM in ChEMBL) and 3) having genetic associations with hypercholesterolemia (for more details, see the Use Cases section).\n\nGenes that satisfied all three requisites above are presented in Figure 5C (CYP7A1, FABP2, LDLR, MYLIP, PCSK9, SREBF2 and STAP1). Among the seven genes we found MYLIP and STAP1. MYLIP is an E3-ubiquitin ligase that degrades LDL receptors in the liver, which are therefore considered to be a potential therapeutic target for dyslipidemia29. Similarly, the STAP1 gene has been recently annotated as a fourth locus associated with autosomal-dominant hypercholesterolemia, and might be a novel target for therapeutic development of hypercholesterolemia30. This result suggests that the new version of TargetMine8 allows us to effectively prioritize target candidate genes in terms of linkage to disease, tractability and competitors. On the other hand, the list includes intractable targets such as PCSK9 and LDLR, indicating the need for improvement of the data and/or the thresholds with which tractable proteins are selected (in this study, ≥50% of protein 3D structures have positive Ensemble scores in DrugEBIlity database).\n\n\nConclusions\n\nThese use cases demonstrate that the updated version of TargetMine8 can be applied in pharmaceutical R&D, from the aspect of understanding the linkage to disease, examining the tractability of targets and prioritizing candidates. The recent update of the Open Targets platform31 also starts to cover “DrugEBIlity” data and protein structural information, suggesting that an integrated resource containing gene-disease associations and tractability information is indispensable for the pharmaceutical R&D. In addition, taking advantage of the features of the InterMine framework, TargetMine8 also facilitates more flexible and more complex queries for advanced users.\n\n\nData availability\n\nThe TargetMine data warehouse is publicly available at https://targetmine.mizuguchilab.org.\n\n\nSoftware availability\n\nSource code available from: https://github.com/chenyian-nibio/targetmine\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.25735658.\n\nLicense: MIT License.", "appendix": "Grant information\n\nThis work was in part supported by JSPS KAKENHI (grant number 17K07268).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank Andrew Myers’ help for improving the quality of writing.\n\n\nReferences\n\nBrown KK, Hann MM, Lakdawala AS, et al.: Approaches to target tractability assessment - a practical perspective. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoscielny G, An P, Carvalho-Silva D, et al.: Open Targets: a platform for therapeutic target identification and validation. Nucleic Acids Res. 2017; 45(D1): D985–D994. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen DT, Mathias S, Bologa C, et al.: Pharos: Collating protein information to shed light on the druggable genome. Nucleic Acids Res. 2017; 45(D1): D995–D1002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSullivan J, Kalderimis A, Butano D, et al.: chenyian-nibio/targetmine: TargetMine v1.8.5.0 (Version v1.8.5.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2573565\n\nKalderimis A, Lyne R, Butano D, et al.: InterMine: extensive web services for modern biology. Nucleic Acids Res. 2014; 42((Web Server issue): W468–472. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalone J, Holloway E, Adamusiak T, et al.: Modeling sample variables with an Experimental Factor Ontology. Bioinformatics. 2010; 26(8): 1112–1118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacArthur J, Bowler E, Cerezo M, et al.: The new NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog). Nucleic Acids Res. 2017; 45(D1): D896–D901. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandrum MJ, Lee JM, Benson M, et al.: ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 2016; 44(D1): D862–868. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandrum MJ, Lee JM, Riley GR, et al.: ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014; 42(Database issue): D980–985. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHigasa K, Miyake N, Yoshimura J, et al.: Human genetic variation database, a reference database of genetic variations in the Japanese population. J Hum Genet. 2016; 61(6): 547–553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagasaki M, Yasuda J, Katsuoka F, et al.: Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals. Nat Commun. 2015; 6: 8018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNHLBI GO Exome Sequencing Project (ESP), S., WA. Vol. 2017.\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\nSudmant PH, Rausch T, Gardner EJ, et al.: An integrated map of structural variation in 2,504 human genomes. Nature. 2015; 526(7571): 75–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbifadel M, Varret M, Rabès JP, et al.: Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet. 2003; 34(2): 154–156. PubMed Abstract \| Publisher Full Text\n\nKiyosue A, Honarpour N, Kurtz C, et al.: A Phase 3 Study of Evolocumab (AMG 145) in Statin-Treated Japanese Patients at High Cardiovascular Risk. Am J Cardiol. 2016; 117(1): 40–47. PubMed Abstract \| Publisher Full Text\n\nTeramoto T, Kobayashi M, Tasaki H, et al.: Efficacy and Safety of Alirocumab in Japanese Patients With Heterozygous Familial Hypercholesterolemia or at High Cardiovascular Risk With Hypercholesterolemia Not Adequately Controlled With Statins　- ODYSSEY JAPAN Randomized Controlled Trial. Circ J. 2016; 80(9): 1980–1987. PubMed Abstract \| Publisher Full Text\n\nClarke L, Fairley S, Zheng-Bradley X, et al.: The international Genome sample resource (IGSR): A worldwide collection of genome variation incorporating the 1000 Genomes Project data. Nucleic Acids Res. 2017; 45(D1): D854–D859. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu ZP, Wang Y: PCSK9 Inhibitors: Novel Therapeutic Strategies for Lowering LDLCholesterol. Mini Rev Med Chem. 2019; 19(2): 165–176. PubMed Abstract \| Publisher Full Text\n\nWei CH, Kao HY, Lu Z: PubTator: a web-based text mining tool for assisting biocuration. Nucleic Acids Res. 2013; 41(Web Server issue): W518–522. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang CP, Tian Y, Zhang M, et al.: IDOL, inducible degrader of low-density lipoprotein receptor, serves as a potential therapeutic target for dyslipidemia. Med Hypotheses. 2016; 86: 138–142. PubMed Abstract \| Publisher Full Text\n\nDay IN: FH4=STAP1. Another gene for familial hypercholesterolemia? Relevance to cascade testing and drug development? Circ Res. 2014; 115(6): 534–536. PubMed Abstract \| Publisher Full Text\n\nCarvalho-Silva D, Pierleoni A, Pignatelli M, et al.: Open Targets Platform: new developments and updates two years on. Nucleic Acids Res. 2019; 47(D1): D1056–D1065. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "45533", "date": "27 Mar 2019", "name": "Rachel Lyne", "expertise": [ "Reviewer Expertise Bioinformatics", "Databases", "Data integration", "data analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe data updates to the already established TargetMine database. The updates allow candidate genes to be checked for genetic evidence for disease and target tractability. The authors clearly demonstrate the utility in integrating the new data through a set of use cases. The sophisticated web interface allows for powerful exploration and advanced querying of the data, making the resource a valuable tool for pharmaceutical research and development. The following minor points should be addressed:\nIn the introduction it is mentioned that additional information and analysis, such as pathway enrichment, are needed to assess aspects of target suitability, but such analysis is not shown in the use-cases. It would be useful to demonstrate features that TargetMine provides that are not available in similar systems such as Open Targets and Pharos. In addition the authors describe how the new data can be used but do not tie this in with the wealth of data already available in TargetMine which could also be used for tractability studies, such as uniprot and interpro protein domains.\n\nTargetMine includes an extensive API but this is not mentioned in the paper. Access to this data through an API could have clear advantages for anyone wishing to set up a workflow or provide a more automated analysis.\n\nThe use case “Gathering and prioritising candidate drug target genes” would be difficult for someone unfamiliar with searching an interMine-based database to reproduce. First, the set of genes (or the filtered set of human genes) should be provided as supplemental material. An overview of a set of queries constructed using the TargetMine query builder is provided. However, to reproduce this set of queries using the query builder is not immediately obvious for a naive user. A more detailed set of screenshots as supplementary material could help, or provision of the set of queries as a series of template searches.\n\nA note on how often the data will be updated should be included.\n\nPlans for further data additions could be included.\n\nThe reference for TargetMine (8) should be updated to contain the correct author list.\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": "45061", "date": "28 Mar 2019", "name": "Anne Hersey", "expertise": [ "Reviewer Expertise Chemoinformatics", "drug discovery", "bioactivity databases" ], "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 produced a nice tool (TargetMine) that can be used for prioritizing targets for drug discovery. They have done this by integrating data from a range of other resources that have the potential to identify the relevance of targets for diseases of interest as well as the likelihood that a small molecule can be found for that target (tractability). The authors have also provided all the code in the form of a github repository and the data model for TargetMine is shown in the article.\n\nThey describe adequately how to use the resource with the aid of a couple of specific use cases that they work through in the article. I have followed through the first example and can obtain the same results as they do. The 2nd example starts from a series of PubMed articles so is less easy to reproduce oneself although the logic is sensible. TargetMine also has links to tutorials that can be used to guide a user through using the resource although I haven’t tried these. In the introduction the authors mention not just using genetic data to assess the relevance of targets to disease but additional information such as pathway analysis although this isn’t exemplified in their use cases; perhaps their use cases could be expanded to include this? In the 2nd use case it would be good to describe at the start why they were prioritizing targets which had no reported inhibitors with IC50s <100nM. I assume this was because they were looking for novelty but this is not explained. To me the 15 targets at the centre of the Venn diagram (figure 5) would also be relevant and tractable targets.\n\nI think it would be useful to be able to link back to the source databases that the information comes from. For example, when I select a UniProt ID I would expect to link back to UniProt itself. Likewise, it would be good to be able to link back to PDB where you can view the 3D structure of the protein. I couldn’t find a way of doing this.\n\nAs the authors mention in their conclusion the Open Targets Platform has also recently started to use the DrugEBIlity algorithms. The authors might be interested to know that this is not currently being further updated and will soon be replaced with an alternative tractability prediction method that will also be free and openly available. A minor but important point: the authors use the words “Ensembl” and “ensemble” to refer to the ensemble of prediction models that are used as one of the measures of tractability. “Ensemble” is the correct word which is very different from “Ensembl” the genome browser (https://www.ensembl.org/). I think it would be useful if the authors could correct this throughout the article.\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/8-233
https://f1000research.com/articles/8-739/v1	24 May 19	{ "type": "Study Protocol", "title": "Protocol for a Japanese nationwide repeated cross-sectional study to assess tobacco and nicotine product use behaviour after market introduction of tobacco heating products (THPs)", "authors": [ "Jason Adamson", "Claudia Kanitscheider", "Krishna Prasad", "Oscar M. Camacho", "Elisabeth Beyerlein", "Yoga Keralapura", "Christopher Proctor", "James Murphy", "Claudia Kanitscheider", "Krishna Prasad", "Oscar M. Camacho", "Elisabeth Beyerlein", "Yoga Keralapura", "Christopher Proctor", "James Murphy" ], "abstract": "Background - In recent years there has been a proliferation of alternative tobacco and nicotine products that reduce consumers’ exposure to harmful substances and therefore have the potential to reduce risk to health. Post-market surveillance enables the evaluation of newly introduced tobacco and nicotine products (aka potentially reduced risk products (PRRPs)) at a population level. This study aims to investigate tobacco and nicotine consumer demographics and discover how people are using these products, and characterise behavioral trends as transitions between tobacco heated products (THPs) and other nicotine products. These behavioural aspects, in conjunction with the intrinsic risk of the product, are essential for assessing the potential health effects and establishing a population risk assessment. Design and methods - This epidemiological cross-sectional study will collect data using a self-administered study instrument from the general Japanese population aged 20 years and older. The targeted sampling size is up to 5,000 participants per study wave. The study addresses the following objectives: estimation of tobacco and nicotine use prevalence; characterisation of product usage by product type; changes in use behaviour in general, with particular emphasis on the introduction of THPs in the time period of one year; risk perceptions of different tobacco products and no tobacco usage; and participant perceived health status and quality of life. Discussion - The description of tobacco and nicotine product use behaviour, the estimation of prevalence data, the measuring of product-specific risk perception and the change of tobacco use behaviour within one year will allow for a comprehensive assessment of the effect of introducing THPs into a market. These data could also be used to inform a system dynamics population model in order to estimate the public health impact of introducing a THP into the Japanese market.", "keywords": [ "Post-market surveillance", "Population studies", "Cross sectional survey", "Tobacco Heating Products (THPs)", "Harm reduction" ], "content": "Introduction\n\nTobacco is used by more than one billion people globally [MacKay et al., 2006], and combustible tobacco is the most common form of tobacco use. Epidemiological data has concluded that smoking significantly increases the risk of diseases such as cardiovascular disease, COPD and lung cancer [US Department of Health and Human Services, 2014]. Tobacco Harm Reduction is a concept described, amongst others, by the Institute of Medicine (IOM) in their monologue Clearing the Smoke [Stratton et al., 2001], which summised that population health could be improved by replacing high-risk combustible tobacco products like cigarettes with innovative lower risk tobacco and nicotine products. This has led to an increase in alternative tobacco and nicotine products that have the potential to reduce exposure to harmful substances and reduce individual health risk. Some have termed such innovations as potentially reduced risk products, or PRRPs, and include electronic cigarettes (e-cigarettes), tobacco heating products (THPs), and oral tobacco products like Scandinavian style snus, amongst others.\n\nThe US Institute of Medicine (IOM) outlined standards for studies assessing the effectiveness of potentially reduced risk products (PRRPs) and implications of their market introduction [IOM, 2012]. Providing data to support the evaluation of behaviour/usage patterns of these new products is one of the important factors in assessing population health risk reduction. For this purpose, not only current tobacco users, but also non-users (never users and former users) must be taken into account. This inclusive approach is pursued to ensure that the population as a whole can benefit from the introduction of PRRPs into a market (not just current combustible tobacco users).\n\nConsumer comprehension of risk perception associated with PRRP use is also crucial for acceptance and continued use, and depends on consumer exposure to product information and peer-to-peer communication [Pepper et al., 2015; Roditis & Halpern-Felsher, 2015]. In addition to risk perception, acceptance and continued use of new products depend on perceived benefits for users. Studies about quality of life (QoL) assessing smoking cessation suggested wellbeing improvements [Piper et al., 2012]. Therefore, it is reasonable for survey based studies to explore the impact of switching to PRRPs on QoL and short-term health effects.\n\nSeveral groups, tobacco industry [Murphy et al., 2017; Smith et al., 2016] and independent [Berman et al., 2015; Farsalinos & Polosa, 2014], have published scientific assessment frameworks which outline the studies that should be conducted and comparator products that should be used to assess the emissions, exposure to harmful and potential harmful constituents (HPHCs) and risks of using PRRPs relative to combustible tobacco smoking. Each of these frameworks describe the requirement of assessing the risk profile of the PRRP at a population level.\n\nBritish American Tobacco’s (BAT’s) approach to population risk profiling for PRRPs is informed by leading national and international public health agencies [CDC, 2012; FDA, 2012; WHO, 2012]. Their recommendations on population based studies allow for the evaluation of the effect of introducing new products on consumer perception, behaviour and health [FDA, 2012]. The overarching objectives of BAT’s surveillance programme are to:\n\nmonitor in-market use-behaviours of tobacco and nicotine consumers in the population\n\nevaluate population perceptions of PRRPs (both users and non users of tobacco and nicotine products)\n\nevaluate the benefits and risks of PRRPs on the health of individuals and on the population as a whole\n\nuse survey generated and market based data to inform a system dynamic population model [Hill & Camacho, 2017] to assess the impact of introducing one or multiple PRRPs on population morbidity and mortality\n\nidentify and collect any adverse events related to BAT’s PRRPs after they are launched in all markets (not just in Japan where this study is conducted)\n\nTobacco heating products (THPs) are just one sub-category of PRRP and contain tobacco that is heated (in a device, or with hot air passing through tobacco) but in contrast to cigarettes is not lit therefore cannot burn, thus there is no combustion [Eaton et al., 2018; Simonavicius et al., 2018; Smith et al., 2016]. The lower temperatures of the THPs heating profile (a maximum of 240°C), compared to combustion temperatures within the lit end of a cigarette (greater than 950°C), results in reduced toxicant emissions [Forster et al., 2018a; Mallock et al., 2018; Schaller et al., 2016] which in turn, results in lower biomarker of exposure (BoE) to HPHCs in humans compared to conventional combustible cigarettes [Gale et al., 2017; Schaller et al., 2016].\n\nThe aforementioned studies have demonstrated that THPs have potential to reduce harm to the individual user, however, the intrinsic risk of the product should also be contextualised within use behaviour patterns after market introduction to enable population risk assessment. Population use patterns would further be linked to consumer risk perception – such as how do non-users, cigarette smokers, solus THP users and dualist (THP and cigarette) users view the risk of THPs – and ultimately how a consumer switch or gradual transition away from cigarette consumption to solus THP use may lead to significant improvements in smoking related health effects and self-percieved quality of life.\n\nJapan is a major market for THPs, in a nation with high smoking prevalence, in particular among men [Levin, 2013]. Of males 15 years and older in Japan in 2015, smoking prevalence (using tobacco daily) was 33.7%; by contrast, prevalence in the UK and US was 19.9% and 19.5% respectively (Tobacco Atlas, 2018). In addition to the individual harm reduction potential already described [Forster et al., 2018a; Gale et al., 2017; Haswell et al., 2018; Jaunky et al., 2018; Mallock et al., 2018; Schaller et al., 2016; Smith et al., 2016], THPs are likely to resonate very well in Japan given the strong cultural values of order, cleanliness, quality and respect for others [Hair et al., 2018], and these could be potential driving factors for Japanese consumers switching from combustible cigarettes to THPs. Thus reduced residual odour on consumers’ clothes and hair [Forster et al., 2018b], reduced teeth staining [Dalrymple et al., 2018], reduced side stream smoke, no ash and improved indoor air quality versus combustible cigarettes (Ichitsubo & Kotaki 2018; Forster et al., 2018b; Mitova et al., 2016; Mottier et al., 2016) may be considered strong additional motivations for some Japanese consumers switching from cigarettes to THPs.\n\nIn Japan, several different tobacco heating products are currently available on the market [Tabuchi et al., 2018]. iQOS from Philip Morris [Smith et al., 2016] was launched countrywide in 2014 after having been tested in selected regions. BAT’s THP glo [Eaton et al., 2018] entered the market at the end of 2016 with a city-launch in Sendai. ploom TECH from Japan Tobacco was introduced in central Tokyo in June 2017 [Reuters, 2018]. All three THPs are referred to in the study instrument (Appendix 1). More recently, Korean Tobacco and Ginseng (KT&G) released their THP Lil in Seoul in November 2017 [Tobacco Reporter, 2017]. Lil was not included in the survey at the time of the pilot study due to its nascent release in South Korea, but may still be accessible to consumers in nighbouring Japan. Additionally there are numerous and diverse THPs emerging from other markets (such as China). Despite this rapidly evolving and innovative product category, this study will allow us to explore the effect of the availability of THPs on the Japanese population as well as to investigate use patterns within this specific consumer product category.\n\nThe following sections of this manuscript will describe the study details, from instrument design and participant selection, through to ethics approval.\n\n\nStudy aim and design\n\nThe aim of this study is to describe the usage patterns of tobacco and nicotine containing products in Japan. This includes the estimation of prevalence rates for overall tobacco use, and by product type as well as usage pattern (solus product use, for example someone who only smokes cigarettes or only uses THPs; dual product use, someone who smokes cigarettes and uses THPs; and multi product use, someone who uses two or more different tobacco and/or nicotine products). In addition, information about frequency and amount of product use is collected as well as intention to quit for cigarette smoking and different THPs. The effect of the market introduction of THPs on the population as a whole will be evaluated for: initiation rates among tobacco non-users and former users; consumer switching rates from cigarettes to THPs, and back again to cigarettes (switch back); initiation of dual use; switching from solus THPs to dual use or solus cigarette smoking (gateway effect) and re-initiation rate of tobacco use among former tobacco users. Risk perception of different tobacco products and health-related QoL are also assessed, as well as smoking related short-term health effects among different tobacco user groups.\n\nThe tobacco use behaviour of the Japanese population will be characterised by the collection of data on a regular basis over a defined time period. This allows the assessment of usage behaviour at specific time points (point prevalence) and also to explore changes on an individual level within the last 12 months. A repeated, nationwide, population-representative cross-sectional survey is chosen to fulfil the study requirements. A pilot study (in Sendai, Tokyo and Osaka only) was implemented to test the envisaged approach in the first year (2018). The results of the pilot study are used to optimise the conduct of the first nationally representative wave commencing in 2019.\n\n\nParticipant selection\n\nA geographically stratified three-stage probability sampling will be applied. The sampling universe comprises all persons aged 20 years and older (which is the legal age to consume tobacco in Japan) living in private households in Japan. Additional inclusion criteria are that the participant is able to speak and read Japanese, and be willing to participate after selection and the information about the study was provided. Pregnant and breast-feeding participants are eligible to participate, as are employees of legal, journalistic, media and tobacco industries; there is no recruitment bias. The only exclusion criterion additional to the legal age of smoking are any persons belonging to the institutionalised population (currently living in prisons, military bases, mental facilities, homes for the aged).\n\nAfter the pilot study (in Sendai (Miyagi Prefecture, Tohoku region), Tokyo (Kanto region) and Osaka (Kinki region)) for subsequent study waves the nation will be stratified based on all 47 prefectures of Japan across nine regions (Chugoku, Hokkaido, Hokuriku, Kanto, Kinki, Kyushu, Shikoku, Tokai, Tohoku). In each of these nine regions, municipalities are stratified into four urbanisation degrees, based on the population of each municipality from the Basic Resident Registration population data: these are major cities (Government-designated metropolitan areas), large cities (>150,000 population, excluding major cities), medium cities (<150,000 population), and small towns.\n\nFirst stage selection is based on the primary sampling units (PSU) which is a street block, selected by stratified random sampling. From the Basic Resident Registration population data (published by the Japanese Ministry of Internal Affairs and Communications (MIC)) within each stratum, 500 PSUs are selected in total proportionately to the population density. PSUs within each stratum are listed in ascending order of the municipality codes, and are selected by a randomly chosen starting number (no greater than the skip interval which is calculated as target population in each stratum divided by number of PSUs in the respective stratum). Second stage selection occurs within each PSU, all of the households are listed in ascending order of the street numbers, using the residential map database (ZENRIN Co Ltd. (ZENRIN)). A household starting number is randomly chosen to select the first household. In total, 50 households per PSU are chosen using regular numeric intervals and are listed on the household list for interviewers. Third stage selection occurs within the selected household and the individual respondent is selected by next birthday method (Salmon & Spicer Nichols, 1983). Only one individual is selected from each household.\n\nThe 20–24 year old population are identified in our sampling universe as a vulnerable group (more susceptible group) as this is most likely where tobacco product initiation takes place; additionally regulators are concerned that PRRPs may appear more attractive and appealing to this age group [FDA, 2012]. As THPs could be considered ‘high tech’ and aspirational products, youth and young adults could be more interested in such product positioning, and therefore this could raise some concern about youth appeal (Hair et al., 2018). Additionally, based on fieldwork experiences in the past, this age group is underrepresented in the applied sampling method, thus in our study the 20–24 year old population are oversampled to ensure robust estimates for this category. The oversampling quota will be assigned to each PSU. Once the target sample size is reached the interviewer will randomly select the household using the same method as for the main sample and uses the quota sampling method for selecting individual respondents aged 20–24 years. Questionnaires from oversampling are specifically marked (by participant ID only, thus participants are unaware of this) to differentiate them from the main sample as different weighting factors will be applied based on selection probability before merging them to one final sample.\n\n\nStudy measures\n\nPrevalence estimates will be calculated for never tobacco users, ever tobacco users, former tobacco users, and current tobacco users, among current tobacco users by product type and for usage patterns (solus product use, dual product use, multi product use). Participants were classified as never, ever, current or former product user depending on if they have ever used the product, used them in the past, or are currently using them and if they smoked at least 100 cigarettes/consumables or used the equivalent amount of tobacco in their lifetime for the following products: manufactured cigarettes/roll your own cigarettes, pipes/kiseru, cigars/cigarillos, THPs (separately for glo, iQOS, Ploom TECH), e-cigarettes and smokeless tobacco products.\n\nFor users of the respective product and partially for former users the following details are collected: duration of product use, when they stopped using the product, frequency and amount of used product, flavour preferences, if applicable, and for current manufactured cigarettes/roll your own cigarettes and THPs their motivation to quit, ever tried to quit, last quit attempt and duration. Motivation to quit tobacco product use will be measured with the Contemplation Ladder [Amodei & Lamb, 2004; Biener & Abrams, 1991]. Tar level and time to first cigarette in the morning was additionally collected for current users of manufactured cigarettes/roll your own cigarettes. For the determination of nicotine dependency the Heaviness of Smoking Index (HSI) [Heatherton et al., 1989] is used. This index consists of two questions considering the amount of smoked cigarettes per day and time to first cigarette smoking in the morning. The composite measure of nicotine dependency is derived from a six-point scale and then categorized as low (0–2), medium (3–4) and high (5–6) dependency.\n\nThe effect of the market introduction of THPs on tobacco use behaviour among tobacco users1 is investigated by collating the following data:\n\nInitiation rate of THPs among combustible tobacco users within the last 12 months\n\nSwitching rate for THPs: percentage of combustible tobacco users who switched from smoking to THPs within the last 12 months\n\nDual use rate (smoking cigarettes and using a THP): percentage of combustible tobacco users who started usage of THPs in parallel to existing smoking behaviour within the last 12 months\n\nPoly-use of THPs: Percentage of current THP users using more than one THP in parallel\n\nSwitching rate from THPs to cigarette smoking (gateway effect): percentage of THP users without smoking history who initiated THP use more than 12 months ago and who switched to solus cigarette smoking within the last 12 months\n\nSwitching rate from THPs to dual use (gateway effect): percentage of THP users without smoking history who initiated use more than 12 months ago and who started smoking in parallel to usage of THPs within the last 12 months\n\nRelapse/switch back rate from THPs to smoking: percentage of THP users with smoking history who initiated use more than 12 months ago and who re-initiated cigarette smoking in parallel to usage of THPs within the last 12 months or who switched back to solus smoking within the last 12 months\n\nCessation rate: Percentage of tobacco users who quit using tobacco products in the last 12 months: overall and by product type\n\nThe effect of the market introduction of THPs on tobacco use behaviour among tobacco non-users2 is investigated by collating the following data:\n\nInitiation rate of THPs among never and former tobacco users within the past 12 months\n\nRe-initiation rate of tobacco products: percentage of former tobacco users who re-initiated tobacco use after a stop period of at least 12 months\n\nPerception of health risk is assessed separately for cigarettes, THPs and no tobacco use in terms of development of lung cancer, respiratory disease and heart disease respectively (indirect measure) with a 7-point scale ranging from “No risk=1” to “Substantial risk=7”. Furthermore, perceptions about the health risk of developing these three diseases from smoking cigarettes compared to using THPs (direct measure) is assessed with the following question: “Compared to continuing to smoke conventional cigarettes, using heated tobacco products has 1-a greater health risk; 2-the same level of health risk; 3-less health risk; 4-no health risk at all; or 5-I don’t know or not sure”. The comparison to cigarettes was chosen as it is the predicate product, and due to its predominant use compared to very small acceptance of other tobacco products, and the ban on e-cigaretes with nicotine containing liquids in Japan.\n\nQoL is assessed among all study participants to explore differences based on tobacco usage behaviour. Global QoL is rated with a single question: “In general, would you say your quality of life is 1-Excellent; 2-Very good; 3-Good; 4-Fair; or 5-Poor”. Health-related QoL was measured separately for physical health, mental health, and overall health using the same 5-point scale. Additionally, 12-month prevalence of smoking-related short-term health effects is measured for 14 conditions releated to teeth, mouth, nose, eyes, skin, lung, athletic ability and addiction to nicotine.\n\nA paper-based questionnaire will be used for data collection (see Extended data (Adamson, 2019)). Prior to fieldwork the questionnaire was forward and back translated to check for accuracy. Thereafter the questionnaire was pre-tested by the fieldwork provider Nippon Research Centre (NRC) with face validation interviews comprising a small sample of participants representing current and never tobacco and nicotine users. The pre-test was implemented to ensure completeness of skip logic as well as to highlight any questions or sections that may have caused uncertainty for the participant. No major issues were identified at the pre-test stage thus the questionnaire was approved for study use. In addition to the above listed study measures, sociodemographic information is collected for all participants as gender, age, region (prefecture), education, employment status, income band, number of persons per residence and marital status. In order to minimize the influence of social desirability of tobacco use on participant response as best possible, and to allow respondents to document honest tobacco product use behaviour, the questionnaire will be self-administered. As with any self-administered questionnaire the quality of the results will be dependent on the honesty of the participant in their responses, as well as potential selection bias, recall bias, and incentive value. The completion of the questionnaire should take approximately 4 to 5 minutes for never users and 15 to 25 minutes for former and current tobacco users depending on the number of different products used. This approach was based on the successful completion of other consumer based studies in Japan.\n\n\nStudy sample size calculation\n\nThis study is descriptive in nature thus no statistical hypotheses are tested. Proportions and rates will be estimated based on the overall population as well as for subgroups of interest like users of tobacco or nicotine-containing products, smokers and users of THPs. In Japan the smoking rate at the time of this study was 19.8%; stratified by gender the proportion of men smoking cigarettes was 31.1% and the proportion of women was 9.5% [Ministry of Health, Labour and Welfare (MHLW) 2016].\n\nTo allow for subgroup analyses and further stratification, a sufficient number of observations per cell is needed [Kish, 1995]. Thus, taking into account smoking prevalence and expecting a male to female ratio of approximately 3:1, a sample size of N=5,000 is chosen.\n\nThe 95% confidence intervals (CIs) are estimated using a conservative exact method proposed by Clopper and Pearson [Clopper & Pearson, 1934]. We accounted for variance inflation due to clustering in k PSUs and m subjects per sampling unit in terms of effective sample size (ESS). The ESS bases on the intra-class correlation coefficient ρ as a relative measure of homogeneity of sampling units and is calculated as ESS = (mk)/(1+ ρ (m-1)) [Killip et al., 2004].\n\nWith N=5,000 respondents randomly sampled, i.e. m=10 subjects per sampling unit, k=500 PSUs and ρ=0.02 (intra-class correlation coefficient) [Adams et al., 2004] the resulting ESS is n=4,237. With an anticipated proportion p=0.20 (20%) of tobacco product users in Japan the absolute precision will be estimated as e=±0.012 on a 95%-confidence level.\n\nFor the pilot study, as only three areas in Japan were selected, with 200 PSUs, a reduced sample size of N=4,000 was considered sufficient. This pilot enables assessment of the envisaged study procedures and to decide if the collected data answer the study objectives during following nationwide waves. An additional sub-sample of 150 subjects for the pilot will be included for the age group 20–24 years as this group could be paramount to understand nicotine and tobacco product use initiation (as previously discussed).\n\n\nData collection process\n\nAfter identification of the target household by sampling the interviewer will visit the household and deliver a postcard containing an explanation of the purpose of the study, a request to participate and contact information (postcard is proprietary to the study fieldwork provider NRC). The interviewer will visit the household on a different day to make a first personal contact. Once the target person in the household has been identified and has agreed to meet/talk to the interviewer, the interviewer will explain the purpose of the study, hand out the Participant Information Letter (proprietary to NRC), and answer questions if necessary. If the target person agrees to participate in the study, the interviewer will provide the questionnaire and request completion. In cases where the participant is able to fill in the questionnaire immediately the interviewer will wait with the participant until the questionnaire is completed. In cases where the participant is not able to fill in the questionnaire immediately, or another household member is met, the interviewer will come back at a later time to collect the completed questionnaire.\n\nIf no member of the household is available the interviewer will put the Participant Information Letter describing the date of visit and the possible date of the next visit in the mail box, and will come back for the maximum of three additional times on different days at different times. If the target person in the household cannot be contacted and the questionnaire cannot be filled in by them, the interviewer proceeds to the next household on their list.\n\nThe interviewer will check the returned data for completeness/correctness and in case discrepancies are detected the interviewer will ask the participant to clarify these issues and/or complete missing parts of the questionnaire. Completed questionnaires are returned in person or per secured postal mail to the fieldwork provider’s facility for data entry. For the completion of the questionnaire all study participants (whether they are a tobacco/nicotine consumer or not) will receive a small incentive in the form of a cash voucher (¥1,000 = ~$9 USD).\n\n\nData management\n\nQuestion routing (skip logic) is applied to guide the participant through the questionnaire. Data of collected paper questionnaires are entered manually by two independent persons into a proprietary electronic data capture (EDC) system (proprietary to Kantar Health GmbH) which is validated according to FDA 21 Code of Federal Regulations Part 11 (FDA 21). All data received will be mapped into a pre-defined format of Clinical Data Interchange Standards Consortium Study Data Tabulation Model (CDISC SDTM). The SDTM will then be transformed into Analysis Data Model (ADaM) data for analysis purposes.\n\n\nData analysis\n\nAll analyses are exploratory and descriptive. Categorical variables will be analysed by frequency tables (total number of observations, number of missing values as additional categories, absolute and relative frequencies). Continuous variables will be reported as summary statistics (total n, number of non-missing and missing values, mean, standard deviation, median, minimum, maximum, and quartiles). For study objectives, 95% confidence intervals will be displayed for the mean of continuous data or frequencies, respectively.\n\nWeighting will be applied to adjust for probabilities of selection of a respondent and non-response with adjustment according to known characteristics of the population. Data will be presented for unweighted and weighted data.\n\n\nEthical study conduct and participant confidentiality\n\nThis study is an epidemiological population survey and is conducted in accordance with the most current versions of the Declaration of Helsinki [World Medical Association, 2013], the guidelines for Good Epidemiological Practice [Public Policy Committee ISoP, 2016] and local laws and regulations. The study is conducted in accordance with the most current version of Marketing Research Guideline and Personal Information Protection Guideline that JMRA (Japan Marketing Research Association) have established. Those guidelines also comply with ICC/ESOMAR codes of conduct.\n\nAt the beginning of April 2018, an independent ethics committee (IEC) in Japan consisting of 9 members (clinical and legal) were informed about the intended data collection and all required documents were provided, including protocol, participant information letter and questionnaire. The IEC from Kitamachi Clinic, Tokyo, reviewed the study documents and approved study conduct on 17th April 2018 before fieldwork commenced (study reference: Repeated Cross-Sectional Study to Assess Tobacco and Nicotine Usage Patterns and Behaviour After Market Introduction of glo in Japan).\n\nDuring fieldwork, potential participants will be informed about the study purpose, their requested tasks, time of involvement, data confidentiality and data protection, incentive (¥1,000 voucher) and contact details in case of questions. By completing the paper questionnaire, the participant implicitly gives consent to participate in this study.\n\nAll participating research parties will ensure compliance with national and international data protection regulations. Every effort will be made to protect participant confidentiality according to Directive 95/46/EC and the Japanese Act on Protection of Personal Information (APPI).\n\n\nAdverse events, product complaints and/or misuse\n\nTo comply with BAT’s product surveillance obligations, there was study monitoring for adverse effects, product complaints, and product misuse for the THP glo (this monitoring occurs for all BAT PRRPs in each market they are sold). The study questionnaire does not actively ask for any product related adverse events (AEs) or product complaints which occurred by using glo. But in case study participants report anything to the interviewer or the interviewer detects any AEs participants experienced during or after the use of glo, this information will be transferred to BAT’s local and central Quality Performance Teams. AEs, complaints or misuse regarding other THPs or cigarettes are out of scope and will not be recorded. In case of glo product complaints the interviewer will advise the participant to call the designated Customer Care Hotline. Frequency and type of product misuse is assessed for glo users in terms of if they ever tried to light a tobacco consumable and smoke it like a conventional cigarette, ever tried to use the device with another different consumable, and/or ever tired to reuse once more an already heated tobacco consumable.\n\n\nLimitations of the research methods\n\nIt could be assumed that in studies on self-reported smoking behaviour that respondents might hesitate to reveal information about their tobacco consumption in the context of a study, and instead provide answers according to social desirability. In order to address this issue, the questionnaire is designed for self-administration to minimise the effect of providing socially desirable responses. Using a non-computer-assisted data collection method in combination with a self-administered questionnaire bears the risk of incomplete and inconsistent data. This will be mitigated by giving clear instructions on how to complete the questionnaire, and by formatting the questionnaire in an intuitive way with clear routing. In addition, finished questionnaires are checked by the interviewer for completeness and consistency of the provided answers during collection at the household.\n\nThe results of this study will be representative for the Japanese population taking into account the local cultural and regulatory environment. Thus, transferability of study outcomes to other countries might be limited.\n\n\nDissemination of information\n\nResults from this study will be shared in publication and at conference(s).\n\n\nStudy status\n\nThe pilot study was fully executed as of April 2019 and the pilot data are currently being drafted into publication. Fieldwork for the first nationwide wave also finished in April 2019; after completion of the data life cycle and reporting the nationwide results will be published.\n\n\nConclusion\n\nPopulation studies, including post-market surveillance and cross sectional surveys such as the study protocol described herein, support the evaluation of the introduction of PRRPs at a population level, in terms of consumer perceptions of product risks, behaviours and impact on public health. Analytical assessments of toxicant emissions (in vapour), pre-clinical assessments and clinical study data have all provided evidence that suggests THPs have the potential to be reduced risk alternatives compared to continued smoking. The post-market assessment of the product gives a more complete understanding of population/public health impact (uptake, switching, quitting, misuse, risk perception, quality of life, etc). Monitoring these behaviours is a regulatory requirement in certain markets, and prevalence data and observations from this study will contribute to new understanding of THP use in Japan, as well as support the areas of epidemiological science for assessing long-term usage of THPs.\n\n\nDeclarations\n\nAll procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. An Independent Ethics Committee from Kitamachi Clinic, Tokyo, reviewed the study documents and approved study conduct on 17th April 2018, prior to fieldwork initiation. Potential participants were informed about the study details before participation, and by agreeing to complete the paper questionnaire thereafter the participant implicitly gave consent to participate in this study.\n\n\nData availability\n\nNo data are associated with this article\n\nThe study postcard and participant information letter are proprietary to Nippon Research Centre (NRC), the fieldwork provider for this study, and therefore cannot be shared. The study questionnaire is openly available from Open Science Framework.\n\nOpen Science Framework: Protocol for a Japanese nationwide repeated cross-sectional study to assess tobacco and nicotine product use behaviour after market introduction of Tobacco Heating Products (THPs). https://doi.org/10.17605/OSF.IO/JECDN (Adamson, 2019).\n\nThis project contains the following extended data:\n\nAppendix_1_Questionnaire.pdf (study questionnaire)", "appendix": "Grant information\n\nThis study was fully funded by British American Tobacco.\n\n\nAcknowledgements\n\nThanks to Madeleine Ashley at BAT R&D for her support with the study questionnaire, and thanks to Jacqueline Cunningham for her support with adverse event reporting and quality performance. Thanks to Shigeki Iwashita at BAT Japan for his local quality review of the study and instrument. Thanks to Andy Hill (Ventana Systems) for his consultation and feedback on the study instrument. Thanks to Stefanie Fülöp at Kantar Germany for protocol and study instrument review. Thanks to Kerstin Becker (ZEG – Berlin Center for Epidemiology and Health Research, Berlin, Germany) for sample size determination, and Martin Pfister (Kantar Germany) for weighting. Final thanks to Hidehiko Otake, Mai Yuasa and colleagues (Kantar Japan), and Kumiko Kondo and colleagues (Nippon Research Center) for their much appreciated services with protocol input and review.\n\n\nFootnotes\n\n1 Tobacco users are participants who were users of any tobacco product 12 months ago.\n\n2 Tobacco non-users are participants who did not use any tobacco products 12 months ago.\n\n\nReferences\n\nAdams G, Gulliford MC, Ukoumunne OC, et al.: Patterns of intra-cluster correlation from primary care research to inform study design and analysis. J Clin Epidemiol. 2004; 57(8): 785–94. 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Reference Source" }	[ { "id": "50426", "date": "24 Jul 2019", "name": "Thomas Verron", "expertise": [ "Reviewer Expertise Data Analytics Research (Statistics", "Datamining)" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written manuscript that provides experimental design on an important topic. However, to enhance reader comprehension, some points should be addressed prior to indexing.\n\nIntroduction This publication described a repeated cross-sectional study, can the author give details about the number of studies (waves) they want to perform and the frequency?\n\nStudy aim and design The participant section should be clarified for a better understanding, e.g.:\nADD: a clear definition of stratum should be mentioned (Prefecture?)\n\nADD: Small towns: population lower than?\n\nADD: The total number of PSUs.\n\nEXPLAIN: How are households ordered in PSU to apply a regular numeric interval?\n\nEXPLAIN: Why do the authors apply the next birthday method, compared to other methods, such as the last birthday method?\nUsing the sampling described, i.e. 500 PSUs * 50 households/PSU * 1 person/household + the young adult sample for the oversampling quota, more than 25,000 will be contacted, and in the study sample size calculation paragraph only 5,000 were interrogated. Can the authors explain this difference?\n\nThere is no information about the type and sources of questionnaires used for this cross-sectional study. For instance, do they use questionnaires developed and validated for the US market and translated to Japanese or developed and validated for the Japanese market?\n\nData availability Can we access to the appendix_1_Questionnaire.pdf (study questionnaire)?\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": "4796", "date": "31 Jul 2019", "name": "Jason Adamson", "role": "Author Response", "response": "Thank you for the review comments. The study was designed to conduct 1-2 waves per year up to three years, or until sufficient data is collected to gain understanding of THP use in Japan and enable calculations of product use transitions. For nationwide waves stratification is by region and urbanization degree. In order to stratify the nation, all municipalities in Japan are classified into nine regions, based on the 47 prefectures. In each of the 9 regions, municipalities are stratified into four urbanization degrees, based on the population of each municipality from the Basic Resident Registration population data. ‘Small towns’ is an official classification: medium cities are “cities less than 150,000 population” and small towns are “towns and villages not classified as cities”. The total number of PSUs selected was 200 for the pilot wave and 500 for the nationwide waves. PSUs are allocated in batches each year and differently for various fieldwork providers. In addition, not every sample point is available for every study as some of them can be blocked due to other studies running in parallel. For the random selection of 500 PSUs, all available PSUs within each stratum are listed in ascending order of the municipality codes assigned to each street block at the time of sampling. The total number of PSUs is 176,815. Participant selection by next birthday method was applied uniformly throughout all study waves; other methods are available and although each may introduce selection bias this would be deemed insignificant. Next birthday method is the usual method employed in Japan. Indeed, to collect responses from 4,000-5,000 respondents, interviewers would need to approach more than 25,000 houses and this is standard procedure for production of contact lists based on the experience of the fieldwork provider. Not all listed households can be reached, not all participants fulfil the inclusion criteria (e.g. able to speak and read Japanese) and not all households are willing to participate. In the fieldwork report after each wave we have a detailed list for non-response/non-participation. The complete study instrument is original and was designed by BAT and Kantar in English with sections of the survey composed of validated tools (in the method section those standard/validated instruments used are mentioned). Though the rest of the instrument is not validated its design follows good practice. The instrument was then translated into Japanese and back translated to ensure accuracy. A small ‘face validity’ assessment was conducted in Japan to ensure the instrument was correct, had accurate skip logic and posed no obvious issues or confusions to participants. A post-hoc instrument validation assessment is also currently being conducted. After each wave, feedback from participants and interviewers is used to improve upon and clarify portions of the instrument. The study instrument (English version) is open access and available on the Open Science Framework here (note that it may not be viewable in certain browsers)." } ] }, { "id": "149120", "date": "28 Sep 2022", "name": "Axel Klein", "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 paper is interesting and makes good points convincingly about the benefits of THR (Tobacco Harm Reduction) with a single country case study. The first thing to strike the reader are significant variations in the quality of writing which suggests that different authors are responsible for different sections. Unfortunately, the flaws in some of the sections detract from the quality of the other sections and the science undergirding the efforts, so it is not ready for indexing as yet.\n\nFor clarity's sake it would have been good to inform readers earlier in the text that nicotine containing e-liquids are prohibited in Japan.\nA couple of additional questions:\nWhat I am not sure about is the use of the term 'potential' here - surely a reduction in toxicant emission and lower biomarkers of exposure reduces harm, not potentially but actually.\n\nThe study of Japan is welcome, but the cultural essentialism smacks of stereotyping even when cast in positive terms: \"resonate very well in Japan given the strong cultural values of order, cleanliness, quality and respect for others [Hair et al., 2018],\". At least, they sit uncomfortably with the above described high incidence of smoking - how did they get away with their stained teeth and inconveniencing others with their smoke, ash and cigarette butts?\nFinally, there is the above mentioned problem with the language in some sections of the text, including a welter of spelling errors and typos make this paper unacceptable right now. A thorough copy edit is required and some sentences need to be reformulated. A few examples:\n\"in their monologue Clearing the Smoke [Stratton et al., 2001],\" = surely monograph not monologue, though it may appear that way.\n\n”Summised”- I am unsure whether the authors mean ‘summarised’ or ‘surmised’ here.\n\n\"Providing data to support the evaluation of behaviour/usage patterns of these new products is one of the important factors in assessing population health risk reduction. For this purpose, not only current tobacco users, but also non-users (never users and former users) must be taken into account\" - garbled - rephrase and simplify.\n\n\"Studies about quality of life (QoL) assessing smoking cessation suggested wellbeing improvements\" - well, what exactly improved wellbeing? Smoking continuation perhaps? It is not clear from this sentence.\n\n\"Several groups, tobacco industry [Murphy et al., 2017; Smith et al., 2016] and independent [Berman et al., 2015; Farsalinos & Polosa, 2014],\" - missing subject\n\n\"is not lit\" - should be 'are'\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/8-739
https://f1000research.com/articles/8-738/v1	24 May 19	{ "type": "Research Article", "title": "Ocular findings in patients with novel A/H1N1 infection: an observational cross-sectional study", "authors": [ "Salil Mehta", "Conrad Rui Vas", "Conrad Rui Vas" ], "abstract": "Background: The systemic findings of novel A (H1N1) influenza have been well documented but data on ocular lesions is scarce. We report the systemic and ophthalmoscopic findings of 14 patients with proven H1N1 infection. Methods: An observational non-interventional retrospective study. During a period of nine years, 14 patients (six female and eight males), were referred for an ophthalmic evaluation. All patients underwent a detailed systemic and ocular evaluation.\n\nResults: Three patients (21.4 %) showed ocular lesions in the form of a unilateral intraretinal hemorrhage. Of these, one patient (7.1%) had a large disc hemorrhage and an area of retinal whitening, consistent with ischemia in the macular area. Conclusions: Physicians dealing with H1N1 infection should be aware of these findings and may include an ocular evaluation as part of their protocol.", "keywords": [ "H1N1", "ocular", "pandemic", "infections" ], "content": "Introduction\n\nNovel A/H1N1 influenza (previously termed as swine flu) is a common infectious disease in India (12,942 infections causing 954 deaths in 20181) and globally2. The respiratory system is primarily affected by infection and extensive data is available for the interaction of A/H1N1 with the respiratory system3. Systemic viral infections (human immunodeficiency virus (HIV), cytomegalovirus, herpes simplex, herpes zoster, chikungunya and West Nile virus) are commonly associated with ocular lesions, usually posterior uveitis, due to their hematogenous dissemination. HIV infection, additionally, shows a classic microvasculopathy (cotton wool spots or hemorrhages) that reflect direct endothelial infection4. However, reports of ocular lesions (infective or vasculopathic) in A/H1N1 infections are limited. Published reports5,6 present single patient findings but aggregate data from multiple cases are not available. This study was intended to ascertain the presence and types of ocular lesions in novel A/H1N1 infections and reports findings from 14 patients.\n\n\nMethods\n\nApproval for retrospective data collection and publication was granted by the institutional ethics committee of Lilavati Hospital and Research Center (approval dated 09/04/2019) The requirement for individual consent was waived.\n\nA retrospective cross-sectional observational study. We evaluated the records of all patients examined in the medical intensive care unit of a medium-sized (320 beds) tertiary referral hospital (Lilavati Hospital and Research Center) from 2015 until 2018. This was done over a month (February 2019). As part of a standard hospital protocol, patients with pyrexia of unknown origin (PUO), at the specific discretion of the admitting physician, undergo a bedside dilated fundus evaluation. A total of 14 patients (six female and eight males, ages ranging from 36–78 years, mean 54.07 years) met the criteria for inclusion (positive tests for novel A/H1N1 infection and having been ophthalmologically evaluated) and their records were analyzed. Of these, 13 (92.8%) were admitted in the intensive care/isolation unit and one (7.1%) chose to be managed on an outpatient basis.\n\nAll patients underwent a detailed evaluation on admission, including a complete blood count (hemoglobin estimation, total and differential white cell counts and platelet counts), blood sugar tests (random on admission, fasting if deemed necessary), liver (serum glutamic-oxaloacetic transaminase (SGOT)/serum glutamic pyruvic transaminase (SGPT)) and renal function tests (serum creatinine and blood urine nitrogen, if needed), urine analysis (routine and microscopy), procalcitonin (PCT) estimation and prothrombin time (PT)/partial thromboplastin time (PTT) studies. Radiological evaluation included chest x-rays and a follow-up chest computed tomogram (CT), at the intensivist’s discretion. An infectious disease evaluation included peripheral blood smears for malarial parasites, rapid NS1 antigen detection for dengue infection and serology for chikungunya (serum IG/IgM) and/or hantavirus infection (serum IgG/IgM estimation). A nasopharyngeal swab was sent for reverse transcriptase-polymerase chain reaction (RT-PCR) for H1N1 infection. Patients who were permitted to receive domiciliary care underwent a slitlamp evaluation, dilated fundus evaluation and intraocular pressure evaluation in the outpatient department.\n\nData were entered into a spreadsheet (Excel for Mac, Microsoft Corp, WA, version 16.16.9) and analyzed with the inbuilt statistical functions.\n\n\nResults\n\nThe presenting complaints of the 14 patients were persistent fever, cough, sore throat and progressively worsening dyspnea for five to seven days prior to admission7. Demographic information and clinical presentations can be found in Table 1. Significant pre-existing diseases included type 2 diabetes mellitus in three patients (21.4%, ages ranging from 74–78 years with a mean of 75.66 years - cases 3, 5 and 14), with additional chronic liver disease in one of these patients (7.4 %, 74-year-old female - case 5).\n\nM, male; F, female; DD, disc diameter; ARDS, adult respiratory distress syndrome.\n\nAll of the patients were febrile, tachypneic and tachycardic. Chest imaging (x-rays and chest scans) revealed extensive bilateral fluffy infiltrates in five patients that were admitted with acute respiratory distress syndrome (35.7% - cases 1, 2, 8, 9 and 11). Other chest imaging findings included unilateral/bilateral patchy infiltrates or consolidation in two patients (21.4 % - cases 4 and 6). Five patients (35.7% - cases 3, 5 and 12–14) had normal chest findings on chest radiology.\n\nThe findings of hematological tests including complete blood count (hemoglobin estimation, total and differential white cell counts) and platelet counts were normal. A bleeding diathesis was ruled out by normal PT/PTT values (11 to 13.5 seconds), in nine (64.2%) of these patients. Liver function tests (SGOT /SGPT) revealed marked hepatic dysfunction in one patient (case 6 – 7.1%) and were normal (SGOT 5–40 U/L, SGPT 7–56 U/L) or borderline elevated in the rest. Renal function tests (serum creatinine and blood urea nitrogen) were normal in all cases (creatinine <1.2 mg%, blood urea nitrogen 5–20 mg%) except three (cases 3,5 and 13 – 21.4%). Random blood sugars on admission were grossly abnormal in one patient (case 9 – 317 mg%) and were normal (< 160mg%) in the rest. Bacterial infection was ruled out, where available, by normal PCT values (<0.5 ng/ml). The results of the tests for malaria, chikungunya, hantavirus and dengue were negative in all patients. Nasopharyngeal swabs were tested using RT-PCR and found to be positive for novel influenza A/H1N1 in all cases. In cases 2, 3, 8–10 and 12–14 (57.1%), the microbiology laboratory was able to confirm the strain as pandemic novel A/H1N1/pdm (2009) strain, but confirmatory evidence of the pdm strain was not available for the rest.\n\nFollowing a dilated fundus evaluation, three patients (21.4 %) showed ocular lesions. These three patients included one male and two females, aged 36–54 years (mean 46.0 years - cases 7, 8 and 9) who demonstrated a unilateral intraretinal hemorrhage. These three patients had platelet counts ranging from 173,000 – 258,000/cu.mm and had no evidence of a bleeding diathesis.\n\nOne patient in this group (case 8) had a large disc hemorrhage and an area of retinal whitening consistent with arterial occlusion and retinal ischemia in the macular area. This patient was a 36-year-old female who rapidly deteriorated with the development of a right cerebral bleed and a right arm radial artery thrombosis that required surgical embolectomy.\n\nThe single patient (case 7) who opted for domiciliary care, underwent a detailed evaluation. This patient’s vision was 6/6, N6 bilaterally with a normal anterior segment examination on slit-lamp evaluation. Dilated fundus evaluation revealed a 1/3-disc diameter (DD) sized retinal hemorrhage in the right eye (Figure 1) and was normal in the left.\n\n\nDiscussion\n\nWe observed ocular lesions in three patients in the form of intraretinal and disc hemorrhages. One patient had disc hemorrhages and macular ischemia, consistent with arterial occlusion/retinal ischemia along with systemic thrombotic events in the form of a cerebral bleed and a radial arterial thrombus. In our patients, thrombocytopenia or a bleeding diathesis did not seem to be the mechanism of these bleeds, thus suggesting an alternate mechanism.\n\nSeveral authors have postulated the virus has a direct cytopathic effect on retinal, neuronal and vascular tissue. Studies of lung tissue have shown similar cytopathic effects on bronchial and alveolar epithelial cells8, as well as intraluminal fibrin thrombi and partial loss of the endothelium in intrapulmonary blood vessels9. We hypothesize that similar patterns of retinal intravascular events, possibly induced directly by the virus or mediated through an immune reaction, may be responsible for our findings.\n\nBunce et al. studied 119 patients with H1N1 infections and were able to identify seven patients with systemic thrombotic events10. Among the likely prothrombotic mechanisms, they identified enhanced platelet activation or endothelial dysfunction, suggesting that the etiopathogenesis of the arterial thrombi may involve endovascular injury or endothelial dysfunction with the release of proinflammatory mediators.\n\nBreker et al. reported a case of a 13-year-old girl with a H1N1 infection who developed bilateral retinal and lateral geniculate body infarction5. They noted an ischemic retinal whitening with inner retinal thickening and hyperreflectivity which they hypothesized was an immune mediated complication. Roesel et al. described a 11-year-old girl with a H1N1 infection, who developed vitritis with massive choroidal exudation which was subsequently confirmed on ultrasound and optical coherence tomography (OCT). Following steroid therapy, the effusion significantly improved6.\n\nThe weaknesses of our study include a referral bias towards critically ill patients and the fact that referral for ophthalmic evaluation was at the discretion of physicians. Hence, this data does not reflect an accurate prevalence of all admitted patients, with a likely skew to a higher frequency of ocular lesions. Virtually all of these patients were isolated or too ill to be moved for a more detailed evaluation including retinal angiography to assess the vascular integrity and patterns of blood flow.\n\nThis small study suggests that hitherto unreported ocular involvement in the form of hemorrhages/retinal ischemia may occur in these patients. Further data is needed to assess their significance, especially as prognostic indicators.\n\n\nData availability\n\nFigshare: H1N1_figshare.xlsx. https://doi.org/10.6084/m9.figshare.80797437\n\nThis project contains the following underlying data:\n\n- H1N1_figshare.xlsx (spreadsheet containing results of tests for each patient)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNational Centers for Disease Control: Seasonal Influenza (H1N1)– State/UT- wise, Year- wise number of cases and death from 2011 to 2018 (till 02nd December, 2018). Accessed 13 December 2018. Reference Source\n\nNovel Swine-Origin Influenza A (H1N1) Virus Investigation Team, Dawood FS, Jain S, et al.: Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med. 2009; 360(25): 2605–2615. PubMed Abstract \| Publisher Full Text\n\nPrasad S, Indhu AJ, Margos RA, et al.: Clinical profile and outcome of H1N1 influenza patients in a tertiary care hospital in Kochi, Kerala. Indian J Respir Care. 2018; 7(2): 97–101. Publisher Full Text\n\nLee JH, Agarwal A, Mahendradas P, et al.: Viral posterior uveitis. Surv Ophthalmol. 2017; 62(4): 404–445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreker DA, Stacey AW, Srinivasan A, et al.: Vision Loss Caused by Retinal and Lateral Geniculate Nucleus Infarction in H1N1 Influenza. J Neuroophthalmol. 2015; 35(3): 265–269. PubMed Abstract \| Publisher Full Text\n\nRoesel M, Heinz C, Heiligenhaus A: H1N1 and uveal effusion syndrome. Ophthalmology. 2010; 117(7): 1467–1467.e1. PubMed Abstract \| Publisher Full Text\n\nMehta S: H1N1_figshare.xlsx. 2019. http://www.doi.org/10.6084/m9.figshare.8079743.v2\n\nMauad T, Hajjar LA, Callegari GD, et al.: Lung pathology in fatal novel human influenza A (H1N1) infection. Am J Respir Crit Care Med. 2010; 181(1): 72–9. PubMed Abstract \| Publisher Full Text\n\nSoto-Abraham MV, Soriano-Rosas J, Díaz-Quiñónez A, et al.: Pathological changes associated with the 2009 H1N1 virus. N Engl J Med. 2009; 361(20): 2001–3. PubMed Abstract \| Publisher Full Text\n\nBunce PE, High SM, Nadjafi M, et al.: Pandemic H1N1 influenza infection and vascular thrombosis. Clin Infect Dis. 2011; 52(2): e14–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49095", "date": "03 Jun 2019", "name": "Reema Bansal", "expertise": [ "Reviewer Expertise Uveitis and Retina" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the abstract, the authors state ‘over a period of nine years’, whereas in the text, the study period mentioned is 2015-2018. Please clarify this. As rightly said in the manuscript, the ocular involvement in H1N1 is very rare. However, the authors have missed discussing the following references, relevant to the disease:\n\nKnapp A. Optic neuritis after influenza, with changes in the spinal fluid. Arch Ophthalmol. 1916;45:247-249.1 Mathur SP. Macular lesion after influenza. Br J Ophthalmol. 1958;42:702.2 Lai CC, Chang YS, Li ML, Chang CM, Huang FC, Tseng SH. Acute anterior uveitis and optic neuritis as ocular complications of influenza A infection in an 11-year-old boy. J Pediatr Ophthalmol Strabismus2011 Jul 6.48 Online:e30-3. doi: 10.3928/01913913-20110628-03.3 Rifkin L, Schaal S. H1N1-associated acute retinitis. Ocul Immunol Inflamm 2012;20:230-232.4\n\nThe manuscript largely discusses the systemic (rather than ocular) findings in details, both in Results as well as Discussion, leading to its suitability as an article for a Medicine journal, rather than Ophthalmology journal. Almost all the references cited by the authors relate to systemic findings in H1N1. Although the authors have discussed the limitations of their study, their findings are not robust enough to have a publication out of this study. None of their patients have been symptomatic for ocular examination. Moreover, the mere finding of an incidental retinal haemorrhage does not merit ophthalmic screening of these critically ill patients.\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? 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": "53508", "date": "23 Sep 2019", "name": "Fehim Esen", "expertise": [ "Reviewer Expertise Ocular inflammation", "uveitis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 ocular findings associated with H1N1 infection. There are 2 cases with asymptomatic retinal hemorrhages and 1 patient with retinal artery occlusion. This report of retinal involvement with H1N1 is new and interesting. The authors also ruled out the possibility of bleeding diathesis in these patients, supporting the possibility these findings are associated directly with H1N1 pathogenesis rather then a result of secondary systemic hematologic changes due to infections in general.\nThere are some issues related with the manuscript:\nThe authors mention that they have collected the patients over 9 years in the abstract. In the methods section, they mention that records of patients between 2015 and 2018 were examined. The dates don’t match.\n\nThe authors report that pre-existing type 2 diabetes in 3 patients among 14 H1N1 patients. However, it is unclear whether the cases that developed retinal lesions had diabetes or hypertension.\n\nThe authors mention that there were 12,942 infections of H1N1 in India last year, but they have only 14 cases from 3 years. A multicenter study with more cases would better describe this disease picture. This can be at least mentioned in 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-738
https://f1000research.com/articles/8-737/v1	24 May 19	{ "type": "Research Article", "title": "Management of infected diabetic wound: a scoping review of guidelines", "authors": [ "Huidi Tchero", "Pauline Kangambega", "Sergiu Fluieraru", "Farid Bekara", "Luc Teot", "Pauline Kangambega", "Sergiu Fluieraru", "Farid Bekara", "Luc Teot" ], "abstract": "Background: Various international guidelines and recommendations are available for management of diabetic foot infections. We present a review of the guidelines and recommendations for management of these infections. Methods: A systematic literature search was conducted through MEDLINE, CENTRAL, EMBASE, LILACS, DARE, and national health bodies. Based on the review of fifteen documents, we present details on the importance of suspecting and diagnosing skin, superficial infections, and bone infections in diabetics. Results: The guidelines recommend classifying the infections based on severity to guide the treatment. While antibiotics have shown the best results, other treatments like hyperbaric oxygen therapy and negative wound pressure have been debated. It is suggested that a team of specialists should be in-charge of managing the infected wounds. Infectious Diseases Society of America (IDSA) 2012 guidelines are widely followed world-over. All guidelines and reviews have consistent suggestions on the assessment of the severity of infection, diagnosis, start, selection, and duration of antibiotic therapy. Conclusions: It is reasonable to conclude that the IDSA 2012 guidelines are commonly followed across the world. There is a consensus among the Australian guidelines, Canadian guidelines, IDSA 2012, National Institute for Health and Care Excellence (NICE) 2015, and International Working Group on the Diabetic Foot (IWGDF) 2016 guidelines on the management of infected wounds for patients with diabetes mellitus.", "keywords": [ "Diabetes mellitus", "Infected wound", "Diabetes complications", "Diabetic ulcers", "Diabetes management guidelines" ], "content": "Introduction\n\nDiabetes mellitus (DM) is one of the major public health issues of this century1. With an increasing life expectancy, the incidence of complications in diabetics is on the rise2,3. Diabetic foot ulcers and infections affect approximately 15% of diabetic patients4,5. An infected foot is a serious complication of diabetes6 and it is a factor in half of all cases of lower extremity amputations7.\n\nVarious guidelines and recommendations by international health bodies and scientific associations, in addition to several systematic reviews and Cochrane reviews, are currently available to guide the selection of the correct treatment modality for infected diabetic foot ulcers/wounds1,8–10.\n\nThere is a general lack of understanding on the infected diabetic wound management guidelines. Further, a comparison of these guidelines is necessary to understand the strengths and weaknesses of these guidelines. Hence, we believe that there was a need to conduct a scoping review to analyze the guidelines that are in practice. The purpose of this scoping review was to study the management practices currently being followed for infected diabetic wounds and present a comparative evaluation of the published guidelines and reviews.\n\n\nMethods\n\nTypes of studies\n\nGuidelines, recommendations or reviews from associations related to diabetes (American Diabetes Association, WHO or any regulatory body) published in English since 2000 and before December 2017 were eligible for inclusion. All the associations that have published guidelines were eligible for inclusion.\n\nTypes of participants\n\nAdults and children with DM\n\nTypes of outcome measures\n\nManagement of infected wounds among patients with DM\n\nAntibiotic therapy\n\nThe following databases were searched on 6th August 2016 using the search terms detailed in Table 1. The databases searched were Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (January 2000 to July 2016), EMBASE, LILACS, The Database of Abstracts of Reviews of Effects (DARE), American Diabetes Association (ADA), European Diabetes Association, WHO, National Institute for Clinical Excellence (NICE) databases and Google Scholar. Clinical trial registries were not searched as the search was for published articles only.\n\nWe searched Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, LILACS, The Database of Abstracts of Reviews of Effects (DARE), American Diabetes Association (ADA) and European Diabetes Association on 6th August 2016.\n\nAll the abstracts and titles of the studies identified by the search were scanned by two authors independently (HT and FB) for relevance according to the inclusion criteria. In the first round, publications were screened using the information in the title and abstract. In the second round, full-texts of the articles identified in the first round were studied to confirm the eligibility. Any differences in opinion about the selection of articles were resolved by a third party (LT).\n\nTwo authors (HT and FB) independently retrieved relevant patients’ and intervention details using standardized data extraction forms using excel sheets. Data were collected under the following headings: Title, Year of publication, Publisher and the following variables of interest: Pathogenesis of diabetic infected wound, Diagnostic guidelines, Diagnosis of osteomyelitis, Antimicrobial treatment, Debridement, Role of other treatment modalities, Requirement for hospitalization, Role of surgery, Wound care, General considerations for management. Disagreements between reviewers, if any, were resolved through discussion to obtain a consensus.\n\nThis report was prepared as per the PRISMA-ScR (Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews) checklist.\n\n\nResults\n\nThe initial search yielded 1025 abstracts, which were screened for potential inclusion in the review. After screening the abstracts, 982 were excluded due to duplication or irrelevance. A total of 15 reports/reviews were included for the scoping review (see Figure 1 for PRISMA flow diagram)11. The initial plan was to compare the management practices for infected diabetic wounds in different countries around the world. However, most of the research and literature were on infected diabetic foot ulcers/wounds. Since diabetic foot ulcers are the most common presentation of diabetic infected wounds, the search results were confined to infected diabetic ulcers. During the search for management practices being followed in different parts of the world, all of the randomized controlled trials (RCTs) that compared different treatment approaches used in the management of infected diabetic wounds were excluded. Moreover, all case reports, case series, observational studies and cohort studies were excluded. Cochrane Reviews were also excluded because each review has taken into consideration one treatment modality. However, systematic reviews and descriptive reviews on different treatment options were considered.\n\nPRISMA flow diagram summarizing identification, screening and inclusion of studies.\n\nIn the next stage, guidelines and recommendations issued by various countries around the world and management review articles were evaluated. Our review included the 200410 and 2012 guidelines of the Infectious Diseases Society of America (IDSA)12, \"Management of Diabetes\" guidelines by the Scottish Intercollegiate13, the International Working Group on the Diabetic Foot (IWGDF) review of systematic reviews14, a clinical update by Australian Diabetes Foot Network8, guidelines for the management of infected ulcers among patients with DM in Portugal15, the Canadian Diabetes Association Clinical Practice Guidelines16, and independent guidelines published by Wounds International in 20137,9,17–22. A summary of these guidelines is presented in Table 2.\n\nAs our scoping review included guidelines, descriptive reviews and systematic reviews, we didn’t use the Assessment of Multiple Systematic Reviews (AMSTAR) tool, which is generally used for quality assessment of the included systematic reviews23. The systematic reviews by Braun et al. (2012) and Peters et al. (2016) had included RCTs after searching PubMed and EMBASE14,17,21. The review by Wukich et al. is a descriptive review without any systematic searches of databases and, of note, the authors had received grants from pharmaceutical companies22. Studies by Joseph and Lipsky, Mansilha and Brandao, and Gemechu et al. were also narrative reviews without any systematic search of the databases7,15,19.\n\nPathogenesis. The guidelines highlighted that the most common causative organisms of diabetic infections are aerobic gram-positive cocci, especially Staphylococcus aureus, including methicillin-resistant strains (MRSA), and Streptococcus spp. (most often Group B). Chronic infections or those occurring after antibiotic treatment are generally polymicrobial, with aerobic gram-negative bacilli and anaerobes. Obligate anaerobes are isolated more commonly from cases of foot ischemia or necrosis. In southern European countries, gram-negative bacilli are more prevalent9,15,16,19,22.\n\nDiagnosis of the diabetic infected wound. All the guidelines, including IDSA 2004, IDSA 2012, IWGDF 2012, and IWGDF 2016 and those by Wounds International Society, recommend that diagnosis of infections should be made on the basis of clinical signs and symptoms9,10,12,14,21. According to the IDSA 2004 guidelines, the diagnosis of infected wounds should be made clinically on the basis of local (and occasionally systemic) signs and symptoms of inflammation12,19. Joseph et al. mentioned that patients should have clinical signs and symptoms of inflammation such as erythema, edema, warmth, tenderness, indurations and/or pain19. The IDSA 2012 guidelines recommend that diagnosis of infections should be based on the presence of at least two classic symptoms or signs of inflammation (erythema, warmth, tenderness, pain or indurations) or purulent secretions. This recommendation was also emphasized by other reviewers7,10,22.\n\nSuspicion/diagnosis of osteomyelitis. The IDSA 2012 guidelines and NICE recommendations (2015) strongly recommend that clinicians should suspect osteomyelitis as a complication of diabetic foot infections (DFIs)10,20. In earlier guidelines, microbiological and laboratory investigations were recommended for the diagnosis of osteomyelitis12. The IDSA 2012, Wounds International guidelines and Australian guidelines suggest that culture and histology findings help with diagnosing osteomyelitis. Due to the unavailability of these tests at many places, clinicians should make a diagnosis in conjunction with clinical, radiographic and laboratory findings8–10. An increase in skin pigmentation may be considered a sign of inflammation and/or infection among patients with pigmented skin8. All guidelines recommend magnetic resonance imaging (MRI) as the best imaging technique to define both soft tissue and bone infections8–10,20. Plain radiography is considered to be less sensitive for DFIs; however, it may be helpful in assessing bone destruction and the presence of gases or foreign bodies9,10,22. As the radiological signs of osteomyelitis are delayed, a normal report resulting from a plain X-ray should be cautiously interpreted9. The IDSA 2012 guidelines strongly recommend that all patients with a new DFI should have a plain radiograph of the affected foot to check whether bones are affected, and whether gases or foreign bodies are present10. Probe-to-bone testing, an inexpensive diagnostic tool, may be helpful in confirming the diagnosis7. Bone samples for culture and histology should be taken after debridement or by bone biopsy10. In addition, white blood cell scanning combined with a radionuclide bone scan can be performed to assist diagnosis9. After the diagnosis of an infected wound and presence or absence of osteomyelitis, it is equally important to classify the severity of the infection, as the treatment choice depends on the severity.\n\nAntimicrobial treatment. Appropriate culture samples should be taken, preferably from soft tissue or purulent secretions, for appropriate selection of antibiotic to be used8,9,20. Tissue specimens or deep swabs should be cultured for both aerobic and anaerobic organisms9. Superficial sampling can miss the true causative organism, thus deep sampling after cleansing or debridement can be helpful9. All guidelines recommend that clinically uninfected ulcers should not be treated with antibiotic therapy. It is strongly recommended that no topical or systemic antibiotic therapy should be given to prevent osteomyelitis, improve wound healing or prevent secondary infection8–10,18,20. Moreover, NICE guideline (2015) also suggested that antibiotic treatment should be started as soon as possible. Culture samples should be taken before the start of the treatment20. NICE guideline (2015) provides wide criteria to choose the appropriate antibiotic and the regimen, such as the severity of the infection, care setting, person’s preference, clinical situation, medical history, microbiological examination, clinical response and cost20. However, tigecycline should not be offered unless other antibiotics are not suitable20. The IDSA 2012, NICE 2015, Wounds International and Scottish guidelines specified that the duration and route of the antibiotic administration should be based on the severity of the disease, presence or absence of bone infection and clinical response to treatment9,10,13,20. In the case of neuroischemic foot ulcer, antibiotics should be chosen carefully as it is more serious than an neuropathic foot ulcer9.\n\nThe IDSA 2012 guidelines stated that the U.S. FDA has approved three antibiotics (ertapenem, linezolid and piperacillin-tazobactum) for the treatment of complicated skin and skin structure infections including DFIs, but not for accompanying osteomyelitis10. However, there is no evidence for the superiority of any particular antibiotic agent, treatment duration or route of administration10,13,14. Wukich et al. mentioned that no one agent or regimen has shown superiority over others; however, those that have demonstrated effectiveness include β lactams (penicillins, cephalosporins), glycopeptides, carbapenems, linezolid, clindamycin and fluoroquinolones22. There is weak evidence to suggest that antibiotic therapy against bone culture leads to higher resolution of bone infection compared to that of empiric therapy10. Also, IDSA 2012 guidelines suggest that antibiotic therapy should be continued only until the resolution of infection10.\n\nEmpiric therapy: IDSA 2004 and 2012 strongly recommend that an empiric antibiotic regimen should be chosen on the basis of the severity of the infection and the likely etiologic agent(s)10,12,19. The guidelines recommend a broad-spectrum antibiotic for severe cases, whereas a narrow spectrum antibiotic should be used for mild cases. The antibiotic agent can be modified following culture reports and antibiotic susceptibility data8–10,16,19,22. The IDSA 2004 guidelines highlighted the importance of escalation and de-escalation regimes depending upon the culture reports12. The local prevalence of MRSA strains should determine the choice of empiric therapy19. Empiric antibiotic therapy against MRSA should be given to patients with a prior history of MRSA infections, or in instances with high local MRSA prevalence colonization, or in cases where the infection is severe9. IDSA 2012 highly recommended that empiric therapy directed at Pseudomonas aeruginosa is usually not required except among those patients with risk factors for Pseudomonas aeruginosa infection10. Recommendations from Wounds International suggest that empiric oral antibiotic therapy against Staphylococcus aureus and β hemolytic Streptococcus are given in the case of mild infections9.\n\nDefinitive therapy: Definitive therapy depends on the culture and sensitivity results of the wound specimen, and on the patient’s clinical response to the empiric regimen10.\n\nMild diabetic foot infections: The guidelines recommend oral antibiotics with a spectrum of activity against gram-positive organisms8–10,20. The treatment should last no longer than 14 days for mild soft tissue infections9,14,15. Wounds International suggests that empiric antibiotic treatment should be changed according to the culture reports. Topical antibiotics can be given along with oral agents. However, topical antibiotics should not be used alone for patients with clinical signs of infection9.\n\nModerate diabetic foot infections: Antibiotic agents against gram positive and gram negative organisms, including anaerobic bacteria, should be administered10,20. The route of administration should depend on the clinical condition and the availability of the antibiotic agents10,20. Recommendations from Wounds International suggest that treatment lasting one to three weeks should be sufficient; however, no specific time is allocated as each decision must be based on the severity and clinical response of the patient9. Other guidelines have also suggested similar periods of 2–3 weeks or 2–4 weeks10,15,20. The empiric antibiotic agent should be changed according to the culture reports or if the signs of inflammation do not improve8,9,13,15,16,19.\n\nSevere diabetic foot infections: Intravenous administration of antibiotic agents against gram-positive and gram-negative organisms, including anaerobic bacteria, should be elicited. The treatment can be switched to the oral route depending upon the culture results and the condition of the patient9,10,20.\n\nOsteomyelitis: Surgical resection or debridement may be required in these cases. Generally, antibiotic therapy must be given parenterally and the duration of antibiotic treatment can last up to six weeks. There is no evidence of superiority of any group of antibiotics or their route of administration over others8–10,16,20.\n\nTopical antibiotic therapy: Although there is no robust evidence to support the use of topical antimicrobials, especially topical antiseptics (such as cadexomeriodine) and silver-based dressings, they are currently being used to decrease the bio-burden of the wound10. However, they may increase the risk of bacterial resistance in addition to causing local adverse effects. The IDSA 2012 guidelines recommended neither the use of topical antimicrobials for most clinically uninfected wounds nor silver-based dressings for clinically infected wounds10. Wounds International suggests that topical antimicrobials may be used alone (but not in patients with clinical signs of infection) or as an adjuvant therapy when there are concerns regarding reduced antibiotic tissue penetration, such as patients with poor vascular supply, and in non-healing wounds with no signs and symptoms of infection, but with increased bacterial bio-burden9. In these cases, topical antimicrobials may prevent the spread of infection to deeper tissues9. Regular monitoring is required to check for improvement and to inform decisions on whether to continue or withdraw treatment9.\n\nDebridement. Wounds International states that mild diabetic infections may require debridement and wounds should be cleaned with saline during the application of every dressing9. The formation of biofilms can be controlled by repeated debridement and cleansing9. IDSA 2012 strongly recommends debridement to remove debris, eschar, or surrounding callus. Sharp debridement methods are considered to be the best; however, other methods such as mechanical, autolytic or larval debridement may be useful in some settings10,18.\n\nRole of other treatment modalities. So far, there has been insufficient data to support the use of other treatment modalities.\n\nGranulocyte colony stimulating factors: There is a weak recommendation for its use as an adjunctive treatment; adding G-CSF did not affect the resolution of infection or the duration of systemic antibiotic therapy10,14,16,17.\n\nHyperbaric oxygen therapy (HBOT): Guidelines and reviews are not in favor of the use of HBOT for ulcer healing, mainly due to the controversial data relating to it14,16,17.\n\nNegative pressure wound therapy (NPWT): This should be considered in patients with active diabetic foot ulcers or postoperative wounds13. Braun et al. stated that there is a moderate level of evidence in favor of using NPWT to heal diabetic foot ulcers17. IWGDF 2016 also suggested that there is a weak evidence in favor of using NPWT in post-operative wounds, although its effectiveness and cost-effectiveness remains unknown21.\n\nTopical antiseptic agents: There is a weak evidence for the use of topical antiseptics such as super oxidized water or iodophor to decrease cellulitis14. The IWGDF 2016 guidelines state that as a result of poor trial designs, it is difficult to draw conclusions in favor of or against the use of topical treatments with antiseptic agents21. The latest IWGDF 2016 recommendations demonstrate little evidence in favor of using honey as an antibiotic agent21. The IDSA 2012 guidelines demonstrate a moderate level of evidence and provide weak recommendations for other modalities such as bioengineered skin equivalents, growth factors, and negative pressure wound therapy10.\n\nRequirement for hospitalization. The IDSA 2012 strongly recommended that patients with a severe infection, some patients with a moderate infection, those who are unable to comply with outpatient treatment, and those with poor response to the therapy should be hospitalized initially10,22.\n\nRole of surgery. Surgical consultation is required for deep abscesses, gas in deeper tissues, extensive bone or joint involvement, gangrene or necrotizing fasciitis7,10,19. Evidence from former systematic reviews demonstrated that early surgery decreases the requirement for amputation significantly in two single center studies14,21.\n\nWound care. Antibiotic therapy is necessary for virtually all infected wounds, but it is often insufficient without appropriate wound care19. Dressings should be chosen according to the nature, depth and size of the ulcer10. Regular monitoring, involving radical and repeated debridement, frequent inspection and bacterial control, are important measures in this regard9.\n\nOff-loading: Off-loading is essential for diabetic foot management. According to a consensus guideline at Journal of the American Podiatric Medical Association, there is a strong evidence that adequate off-loading increases the likelihood of diabetic foot ulcer (DFU) healing24. Nonremovable casts or fixed ankle walking braces are currently perceived as optimum off-loading modalities. However, a gap still exists between what the evidence suggests and what is being performed in clinical practice.\n\nGeneral considerations for management.\n\nRole of teams and specialists: All guidelines stressed the importance of having teams of specialists treating diabetic infected wounds8–10,22. Specialists should be sought if the attending physician is not familiar with the techniques of wound care10. Diabetic foot care teams can include (or should have ready access to) specialists in various fields10. It is strongly recommended to consult a vascular surgeon for revascularization for patients with evidence of ischemia of the infected limb10. Similarly, Wounds International suggested surgical consultation for rapidly deteriorating wounds that do not respond to antibiotic therapy9. Glycemic control is also important during the management of diabetic infected wounds as the correction of hyperglycemia may lead to a favorable response8–10,22. Lipid and blood pressure levels should be within control, and smoking cessation should be advised8.\n\nPatient education: Being the primary care-givers for their own feet, patients should be aware of the risk factors that could predispose to or worsen DFIs and the appropriate care and management behaviors. Former studies showed that patient education programs could be of substantial benefit in reducing the incidence of DFUs and improving self-care practices25. Patients should be taught to examine their feet daily and report any abnormality to their physician, trim toenails with a safety clipper and wear offloading casts. Moreover, patients should be aware of the importance of exercise, smoking cessation for smokers and compliance to diabetes control instructions26,27.\n\nAmputation: Although not a preferred treatment approach, amputation may be required in certain situations, such as life-threatening foot infections that cannot be managed by other measures, non-healing ulcers with a disease burden higher than expected after amputation or where ischemic rest pain cannot be managed by analgesia or revascularization9,10,22.\n\n\nDiscussion\n\nThis scoping review aimed to compare the management practices currently being followed in different parts of the world to treat diabetic infected wounds. As described in the results section, research is ongoing to decide appropriate management of diabetic infected wounds. The literature search identified the guidelines/recommendations and systematic reviews published on the management of diabetic infected wounds from 2000 to August 2016. The aim to consider the global practices was achieved, as recommendations from North America10,16,22, Europe13,15,20, Australia8, and International scientific societies9,18 were included.\n\nOur review highlights that the guidelines across the world provided similar recommendations for the management of infected diabetic wounds. The first stage of suspecting and diagnosing infections was emphasized by all the guidelines. The IDSA 2004 guidelines recommended diagnosis based on the presence of clinical symptoms and signs of local inflammation; however, the IDSA 2012 guidelines recommended diagnosis based on the presence of two clinical symptoms and signs of local inflammation10,12. There is a consensus among the guidelines on the requirement to suspect and quickly diagnose osteomyelitis10,18,21. MRI was established as the best diagnostic method, while plain X-ray should not be considered for diagnosis9,10,20.\n\nAll the included reviews and guidelines have concluded that most acute infections are caused by gram-positive cocci, S. aureus and Streptococci, and that gram-negative cocci or anaerobes may be involved, as infections are generally polymicrobial8–10,15. The IDSA 2012 clinical practice guideline has suggested the use of any of three antimicrobials (ertapenem, linezolid, and piperacillin-tazobactum) for diabetic infected wounds; however, there is a consensus on the non-superiority of any one antibiotic agent over the other two9,10,13. Similarly, there is a consensus on the importance of local protocols, the prevalence of MRSA, and the severity of the wound as important deciding factors while selecting the appropriate antibiotic therapy9,10. NICE 2015 further highlighted the importance of cost when choosing the antibiotic agent20. The severity classification of IDSA 2012 has been accepted universally10. There are similar recommendations for the choice of empiric therapy and the duration of the antibiotic therapy across all included reviews.\n\nThe guidelines did not suggest anti-microbial use for clinically uninfected wounds8–10,20. Other similar recommendations include the importance of regular monitoring and the crucial role of multidisciplinary teams consisting of microbiologists, infectious disease specialists, surgeons and medical specialists8,10,15.\n\nThe guidelines and reviews provided strong recommendations on the previously mentioned modalities; however, there is weak evidence to support some routinely followed treatment practices. With respect to hyperbaric oxygen therapy, the latest guidelines provide weak evidence to support the use of alternative modes such as hyperbaric oxygen or NPWT8–10,20,22. However, this modality requires further exploration and research. A retrospective study of patients with DM with hand infections demonstrated that the addition of HBOT to standard therapies is safe due to its anti-infective effects28.\n\nRegarding wound care, the analysis demonstrates that it is equally as vital as the use of antibiotic agents. However, again, there is a lack of evidence in favor of the many available wound care modalities. At the same time as IDSA and NICE guidelines were released, various systematic reviews have sought to evaluate the options of wound care. Alginate dressings, foam dressings and hydrocolloid dressings were not found to promote the healing of diabetic foot ulcers any better than other alternative dressings. The reviews concluded that the decision-makers may consider other aspects such as cost and wound management properties when selecting the dressing type2,29. Another systematic review by Cochrane showed that NPWT may be an effective treatment to heal debrided foot ulcers and post-operative amputation wounds; however, the studies included could be at risk of bias30.\n\nWhile conducting this scoping review, all English guidelines published by the previously mentioned associations from around the world since 2000 were included. Strengths of this scoping review are the inclusion of recommendations from different corners of the world and an extensive search of various databases. However, the inclusion of only English language published reviews limited the search. It was not possible to calculate the quality of all the articles included as some were guidelines that could not be assessed by the scales available.\n\n\nConclusion\n\nIt is reasonable to conclude that the IDSA 2012 guidelines are commonly followed across the world. There is a consensus among the Australian guidelines, Canadian guidelines, IDSA 2012, NICE 2015 and IWGDF 2016 guidelines on the management of infected wounds for patients with DM. Due to the lack of evidence, the therapeutic status of treatment options like hydrocolloid gels, NPWT, hyperbaric oxygen and aligate dressings could not be ascertained. There is a need to generate stronger evidence regarding the commonly used methods in the treatment of diabetic wound infections.\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\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAtlas ID: International Diabetes Federation, Brussels, 2015. 2015; [Last accessed: 5 March 2014].\n\nDumville JC, Deshpande S, O'Meara S, et al.: Foam dressings for healing diabetic foot ulcers. Cochrane Database Syst Rev. 2011; (9): CD009111. PubMed Abstract \| Publisher Full Text\n\nGregg EW, Sorlie P, Paulose-Ram R, et al.: Prevalence of lower-extremity disease in the US adult population >=40 years of age with and without diabetes: 1999-2000 national health and nutrition examination survey. Diabetes Care. 2004; 27(7): 1591–1597. PubMed Abstract \| Publisher Full Text\n\nSingh N, Armstrong DG, Lipsky BA: Preventing foot ulcers in patients with diabetes. JAMA. 2005; 293(2): 217–228. PubMed Abstract \| Publisher Full Text\n\nBuchberger B, Follmann M, Freyer D, et al.: The importance of growth factors for the treatment of chronic wounds in the case of diabetic foot ulcers. GMS Health Technol Assess. 2010; 6: Doc12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNelson EA, O'Meara S, Craig D, et al.: A series of systematic reviews to inform a decision analysis for sampling and treating infected diabetic foot ulcers. Health Technol Assess. 2006; 10(12): iii–iv, ix–x, 1–221. PubMed Abstract \| Publisher Full Text\n\nGemechu FW, Seemant F, Curley CA: Diabetic foot infections. Am Fam Physician. 2013; 88(3): 177–84. PubMed Abstract\n\nBergin SM, Gurr JM, Allard BP, et al.: Australian Diabetes Foot Network: management of diabetes-related foot ulceration - a clinical update. Med J Aust. 2012; 197(4): 226–9. PubMed Abstract \| Publisher Full Text\n\nChadwick P, Edmonds M, McCardle J, et al.: International best practice guidelines: wound management in diabetic foot ulcers. Wounds Int. 2013; 4: 1–20.\n\nLipsky BA, Berendt AR, Cornia PB, et al.: 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clin Infect Dis. 2012; 54(12): e132–173. PubMed Abstract \| Publisher Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLipsky BA, Berendt AR, Deery HG, et al.: Diagnosis and treatment of diabetic foot infections. Clin Infect Dis. 2004; 39(7): 885–910. PubMed Abstract \| Publisher Full Text\n\nNetwork SIG: Management of diabetes: A national clinical guideline. 2010. Reference Source\n\nPeters EJ, Lipsky BA, Berendt AR, et al.: A systematic review of the effectiveness of interventions in the management of infection in the diabetic foot. Diabetes Metab Res Rev. 2012; 28 Suppl 1: 142–162. PubMed Abstract \| Publisher Full Text\n\nMansilha A, Brandão D: Guidelines for treatment of patients with diabetes and infected ulcers. J Cardiovasc Surg (Torino). 2013; 54(1 Suppl 1): 193–200. PubMed Abstract\n\nCanadian Diabetes Association Clinical Practice Guidelines Expert Committee, Bowering K, Embil JM: Foot care. Can J Diabetes. 2013; 37 Suppl 1: S145–149. PubMed Abstract \| Publisher Full Text\n\nBraun LR, Fisk WA, Lev-Tov H, et al.: Diabetic foot ulcer: an evidence-based treatment update. Am J Clin Dermatol. 2014; 15(3): 267–281. PubMed Abstract \| Publisher Full Text\n\nGame FL, Attinger C, Hartemann A, et al.: IWGDF guidance on use of interventions to enhance the healing of chronic ulcers of the foot in diabetes. Diabetes Metab Res Rev. 2016; 32 Suppl 1: 75–83. PubMed Abstract \| Publisher Full Text\n\nJoseph WS, Lipsky BA: Medical therapy of diabetic foot infections. J Vasc Surg. 2010; 52(3 Suppl): 67S–71S. PubMed Abstract \| Publisher Full Text\n\nNICE: Diabetic Foot Problems: Prevention and Management. 2015. PubMed Abstract\n\nPeters EJ, Lipsky BA, Aragón-Sánchez J, et al.: Interventions in the management of infection in the foot in diabetes: a systematic review. Diabetes Metab Res Rev. 2016; 32 Suppl 1: 145–153. PubMed Abstract \| Publisher Full Text\n\nWukich DK, Armstrong DG, Attinger CE, et al.: Inpatient management of diabetic foot disorders: a clinical guide. Diabetes Care. 2013; 36(9): 2862–2871. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShea BJ, Grimshaw JM, Wells GA, et al.: Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews. BMC Med Res Methodol. 2007; 7(1): 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSnyder RJ, Frykberg RG, Rogers LC, et al.: The management of diabetic foot ulcers through optimal off-loading: building consensus guidelines and practical recommendations to improve outcomes. J Am Podiatr Med Assoc. 2014; 104(6): 555–567. PubMed Abstract \| Publisher Full Text\n\nLincoln NB, Radford KA, Game FL, et al.: Education for secondary prevention of foot ulcers in people with diabetes: a randomised controlled trial. Diabetologia. 2008; 51(11): 1954–61. PubMed Abstract \| Publisher Full Text\n\nEkore RI, Ajayi IO, Arije A, et al.: Knowledge of and attitude to foot care amongst Type 2 diabetes patients attending a university-based primary care clinic in Nigeria. Afr J Prim Health Care Fam Med. 2010; 2(1): 175. Publisher Full Text \| Free Full Text\n\nGoie TT, Naidoo M: Awareness of diabetic foot disease amongst patients with type 2 diabetes mellitus attending the chronic outpatients department at a regional hospital in Durban, South Africa. Afr J Prim Health Care Fam Med. 2016; 8(1): e1–e8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAydin F, Kaya A, Savran A, et al.: Diabetic hand infections and hyperbaric oxygen therapy. Acta Orthop Traumatol Turc. 2014; 48(6): 649–654. PubMed Abstract \| Publisher Full Text\n\nDumville JC, Deshpande S, O'Meara S, et al.: Hydrocolloid dressings for healing diabetic foot ulcers. Cochrane Database Syst Rev. 2013; (8): CD009099. PubMed Abstract \| Publisher Full Text\n\nDumville JC, Hinchliffe RJ, Cullum N, et al.: Negative pressure wound therapy for treating foot wounds in people with diabetes mellitus. Cochrane Database Syst Rev. 2013; (10): CD010318. PubMed Abstract \| Publisher Full Text" }	[ { "id": "63395", "date": "22 May 2020", "name": "Nousheen Bibi", "expertise": [ "Reviewer Expertise My area of research is infectious diseases", "neurology", "and cancer biology. As a bioinformatician", "I have expertise in data collection and analysis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the research article \"Management of infected diabetic wound: a scoping review of guidelines\" Huidi et al. have conducted a study and concluded that all guidelines and reviews have consistent suggestions on the assessment of the severity of infection, diagnosis, start, selection, and duration of antibiotic therapy. Overall the research work is well organized and presented. The methodology is sound and can be replicable. The author has concluded due to the lack of evidence, the therapeutic status of treatment options like hydrocolloid gels, NPWT, hyperbaric oxygen, and alligate dressings could not be ascertained. There is a need to generate stronger evidence regarding the commonly used methods in the treatment of diabetic wound infections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "63468", "date": "01 Jun 2020", "name": "Davoud Dastan", "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 review by Huidi Tchero et al. reports ‘Management of infected diabetic wound: a scoping review of guidelines’. The authors claimed that the results are encouraging to develop a plan for diabetic wound and eco-friendly approach. This article signifies the importance of suspecting and diagnosing skin, superficial infections, and bone infections in diabetics. The suggested guidelines seem consistent with the suggestions on the assessment of the severity of infection, diagnosis, start, selection, and duration of antibiotic therapy. The authors further claimed that the IDSA 2012 guidelines are commonly followed across the world and there is a consensus among the Australian guidelines, Canadian guidelines, IDSA 2012, NICE 2015 and IWGDF 2016 guidelines on the management of infected wounds for patients with DM. Due to the lack of evidence, the therapeutic status of treatment options like hydrocolloid gels, NPWT, hyperbaric oxygen and aligate dressings could not be ascertained. There is a need to generate stronger evidence regarding the commonly used methods in the treatment of diabetic wound infections. The authors may refer to this latest literature to enhance their review; Aljerf et al. (20191).\nThe manuscript has however some grammatical mistakes which need to be improved. As a whole, the topic is interesting. The manuscript is well organized and the authors systematically addressed the issues. I recommend indexing of this article after minor revisions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "63740", "date": "01 Jun 2020", "name": "Muhammad Bilal Tahir", "expertise": [ "Reviewer Expertise Infection diseases", "Diabetic patients", "Nanotechnology in 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\nThe authors comprehensively discussed the diabetic patients, their complications, and recommended the future guidelines. A thorough study was conducted using MEDLINE, CENTRAL, EMBASE, LILACS, DARE, and national health bodies. Additionally, importance of suspecting and diagnosing skin, superficial infections, and bone infections in diabetics have been studied. I strongly recommend it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/8-737
https://f1000research.com/articles/8-727/v1	23 May 19	{ "type": "Research Note", "title": "The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K", "authors": [ "Kurt Tobler", "Claudia Senn", "Elisabeth M. Schraner", "Mathias Ackermann", "Cornel Fraefel", "Peter Wild", "Kurt Tobler", "Claudia Senn", "Elisabeth M. Schraner", "Mathias Ackermann", "Cornel Fraefel" ], "abstract": "Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants (HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. Methods: We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. Results: CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Conclusions: Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.", "keywords": [ "HSV-1", "envelopment", "nuclear membranes", "Golgi membranes", "gK", "immunogoldlabeling", "immunofluorescence", "TEM" ], "content": "Introduction\n\nCapsids of herpes simplex virus 1 (HSV-1) are assembled in host cell nuclei, transported to the nuclear periphery and translocated to the perinuclear space (PNS) or into the cytosol (Roizman et al., 2007). The HSV-1 Us3 kinase phosphorylates numerous viral and cellular substrates (Kato & Kawaguchi, 2018). It is involved in nucleus-to-cytoplasm capsid translocation (Kato et al., 2009; Mou et al., 2009; Reynolds et al., 2002; Ryckman & Roller, 2004; Wisner et al., 2009), in regulation of phospholipid synthesis induced by HSV-1 (Sutter et al., 2012; Wild et al., 2012), and in blocking apoptosis induced by HSV-1 (Galvan & Roizman, 1998; Goodkin et al., 2004; Leopardi et al., 1997). The Golgi complex plays a significant role in apoptosis (Cheng et al., 2010; Hicks & Machamer, 2005), in membrane biosynthesis together with the endoplasmic reticulum (Smirle et al., 2013), and in HSV-1 envelopment (Roizman et al., 2014). Glycoprotein K (gK) is a hydrophobic transmembrane protein of the viral envelope (Mo & Holland, 1997) encoded by the UL53 gene (Bond & Person, 1984; Hutchinson et al., 1992). gK is essential for efficient envelopment by Golgi membranes (Baines et al., 1991; Melancon et al., 2007) and for production of infectious progeny virus (Chouljenko et al., 2012). HSV-1ΔUs3 virions accumulate in the PNS. Nuclear and Golgi membranes become severely altered by insertion of proteins of unknown nature (Wild et al., 2015). The essentiality of gK in envelopment by Golgi membranes prompted us to identify gK in cells infected with HSV-1ΔUs3. Because of the hydrophobicity of gK, we constructed a HSV-1ΔUs3 with a hemagglutinin (HA) tag at gK, CK177ΔUs3gK-HA. Immunogold labeling shows that gK localizes on amorphous protein structures, nuclear and Golgi membranes, and, importantly, on virions in the PNS.\n\n\nMethods\n\nVero cells (European Collection of Cell Cultures, ECACC, 84113001) were grown in Dulbecco’s modified minimal essential medium (DMEM; 31885-023; Gibco, Bethesda, MD, USA) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) (Anti-Anti, 15240-062, Gibco) and 10% fetal bovine serum (FBS; 2-01F10-I, Bio Concept, Allschwil, Switzerland). The Us3-deletion mutant R7041ΔUs3 was kindly provided by Bernard Roizman (The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, Illinois, USA). Wild-type (wtHSV-1) strain F (Ejercito et al., J. Gen. Virol. 2:357–364, 1968), R7041ΔUs3 were propagated in Vero cells.\n\nRecombineering of pYEbac102 (Tanaka et al., 2003) was done in two steps in Escherichia coli strain SW102 (Warming et al., 2005) First, a galK expression cassette was amplified by PCR (Phusion R, High-Fidelity DNA Polymerase; M0530L, BioLabs. Ipswich, MA, USA, according to manufacturer’s recommendations) with homology arms flanking UL53 using the following primers: primer forward, 5’- tct tcg gtg cca gtc cgc tgc acc gat gta ttt acg cgg tac gcc cca ccg CCT GTT GAC AAT TAA TCA TCG GCA -3’; primer reverse, 5’- gtt tcc aat ttg cat atg ccg tta cgg ttt ccg ccg gcc tgg atg tga cgt TCA GCA CTG TCC TGC TCC TT -3’ (binding sequences in capitals). This amplimer was DpnI treated to remove template DNA, purified, and electroporated into competent and induced E. coli strain SW102 carrying pYEbac102 (Tanaka et al., 2003). Recombinant colonies were selected for growth on galactose plates. The BAC DNA carrying the HSV genomic sequence with deleted UL53 was designated pYEbac102ΔUL53. Second, a kanamycin resistant cassette was amplified by PCR on pBSrspL (Genes Bridges, Dresden, Germany; kanamycin resistant cassette) with the following primers for Us3rpsL: primer forward, 5’-ctt ccc aca cca cac cac cca gcg agg ccg agc gcc tgt gtc atc tgc aGG GCC TGG TGA TGA TGG CGG GAT CG-3’, primer reverse 5’-aga tca cca gac cgg cgc tcc aaa tgt cga cgg tcg tgg tat acg gat ccT CAG AAG AAC TCG TCA AGA AGG-3’ (binding sequences in capitals). The primers were chosen to yield the same deletion of Us3 as described by Purves et al. (1987). This amplimer was DpnI-treated, purified, and electroporated into competent and induced E. coli strain SW102 carrying pYEbac102ΔUL53. Recombinant colonies were selected for growth on kanamycin plates. Finally, fHSVgKgalKΔUS3, pCS177 (HA tagged gK and flanking sequences, see below) and pCMV-Cre (Cre recombinase under CMV promoter) DNA was mixed and co-transfected into Vero cells using Lipofectamine 2000 (11668027, Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s recommendations. Rescued progeny virus with deleted Us3 and BAC backbone but HA-tagged gK, designated CK177ΔUs3gK-HA, was propagated in Vero cells.\n\nPlasmid pCS177 containing the UL53 gene with a carboxy terminal HA tag and flanking sequence to UL53 that extend 0.3 kbp upstream and 0.4 kbp downstream of the UL53 gene was constructed as follows. First, downstream sequence of UL53 was amplified by PCR from wtHSV-1 genome (primer forward, 5’- gat ctc tag acg tca cat cca ggc cgg cgg aa - 3’; primer reverse, 5’- gat cga gct cag gCC TCC GGC ACA GAC AAG GAC CAA T -3’; HSV-1 sequences in capitals). The resulting PCR product was digested with SacI and XbaI and cloned into the SacI and XbaI sites in pBluescript II KS(+), resulting in plasmid pCS176. Second, the UL53 gene with its upstream flanking sequence was amplified by PCR from wtHSV-1 using a reverse primer containing nucleotides of HA tag (primer forward, 5’- gat caa gct tag gcc tgg gtc ggt aca acg tac agc cgg at - 3’; primer reverse, 5’- gat ctc tag aTC Acc atg gag cat aat ctg gaa cat cat atg gat aTA CAT CAA ACA GGC GCC TCT gga -3’; HSV-1 sequences in capitals). Following PCR, the DNA product was digested with HindIII and XbaI and inserted into these sites in pCS176, resulting in plasmid pCS177. The StuI fragment of pCS177 containing UL53-HA gene with flanking sequence was co-transfected together with pYEbac102ΔUL53 and pCMV-Cre into Vero cells. gK-HA expression was identified using indirect immunofluorescence and one virus stock expressing gK-HA was designated CK177gK-HA.\n\nVero cells were grown for 2 days on cover slips (Assistent, Sondheim, Germany) for immunofluorescence, on sapphire disks (100.00174, Bruegger, Minusion, Switzerland) placed in 6 well plates for TEM, or in 75 cm2 cell culture flasks for immunogoldlabeling, for 2 days prior to inoculation with CK177ΔUs3gK-HA, R7041ΔUs3, CK177gK-HA or wtHSV-1 at a multiplicity of infection (MOI) of 1 plaque-forming unit (pfu)/ml.\n\nCells on sapphire disks were frozen 16 to 24 hpi in a high-pressure freezer (HPM010; BAL-TEC Inc., Balzers, Liechtenstein) and prepared for sectioning as described in detail previously (Wild et al., 2018; Wild et al., 2002). Cells were analyzed in a transmission electron microscope (CM12; FEI, Eindhoven, The Netherlands) equipped with a CCD camera (Ultrascan 1000; Gatan, Pleasanton, CA, USA).\n\nCells inoculated with CK177ΔUs3gK-HA or CK177gK-HA at MOI 1 were incubated for 20 h, briefly washed with PBS, fixed with 2% formaldehyde for 25 min at room temperature, washed with cold PBS, permeabilized with 0.1% Triton-X-100 at room temperature for 7 min, and blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST). Cells were labeled with mouse monoclonal HA-probe antibodies (1:500) (SC-7392, Santa Cruz Biotechnology, Dallas, TX, USA), followed by anti-mouse Alexa 488-conjugated secondary antibodies (1:500) (A1101; Thermo Fisher) as well as with polyclonal antibodies (1:500) raised in rabbits against Us3 (kindly provided by Bernard Roizman) followed by anti-rabbit Alexa 594-conjugated secondary antibodies (1:500) (A11037; Thermo Fisher). After staining nuclei with 4',6-diamidino-2-phenylindol (DAPI; Roche, Mannheim, Germany), cells were embedded in glycergel mounting media (C0563; Dako North America, Carpinteria, CA, USA) and 25 mg/ml DABCO (1,4-diazabicyclo [2.2.2] octane; 33480, Fluka, Buchs, Switzerland) and analyzed using a confocal laser scanning microscope (SP2, Leica, Wetzlar, Germany).\n\nInoculated cells were harvested at 20 hpi and prepared according to (Tokuyasu, 1973; Tokuyasu, 1980). Cells fixed with 4% formaldehyde in 0.1 M Na-phosphate, pH 7.4, for 2 h at room temperature were scraped from the flasks, washed three times with 0.1 M Na-phosphate by centrifugation at 13,000g for 30 s, and pelleted in 12% gelatine by centrifugation at 13,000g for 3 min at 37°C. After gelation at 4°C, 1 mm3 blocks were immersed overnight in 2.3 M sucrose constantly rotating at 4°C. The infiltrated blocks were mounted on specimen holders, frozen by plunging into liquid nitrogen, and placed into the cryo-microtome (UC6, Leica, Vienna, Austria) at -120°C. Ultra-thin sections of 80–100 nm were collected on carbon-coated formvar films mounted on hexagonal 100 mesh/inch copper grids. Sections were washed by floating on several drops of buffer and blocking solutions prior to routine labeling procedure (Slot et al., 1991) with primary antibodies (1:30) against HA (SC-805, Santa Cruz Biotechnology), and secondary anti-rabbit antibodies (1:30) coupled to 12 nm colloidal gold (111-205-144, Jackson ImmunoResearch, West Grove, PA, USA). After labeling, sections were washed on distilled water droplets, stained by immersing in a mixture of 1.8% methyl cellulose and 0.4% uranyl acetate (Griffiths et al., 1983) for 10 min, and dried for imaging by TEM (Philips, CM12). For controls, primary antibodies were omitted, and labeling was also performed in wtHSV-1 infected cells.\n\n\nResults\n\nInfection with HSV-1ΔUs3 induces formation of folds and invagination of nuclear membranes associated with accumulation of virions (Poon et al., 2006; Reynolds et al., 2002; Wisner et al., 2009). Infection with CK177ΔUs3gK-HA, a similar mutant lacking Us3 but equipped with an epitope-tagged gK, also induced multiple folds and invaginations of nuclear membranes containing virions. Moreover, homogenous, proteinaceous structures occurred within nuclei and cytoplasm of CK177ΔUs3gK-HA (Figure 1) and R7041ΔUs3 (Figure 2A) infected cells. Golgi complexes consisted of multiple stacks comprising thick electron dense membranes especially at the trans face (Figure 3) similarly as described in R7041ΔUs3 (Figure 2B) infections (Wild et al., 2015). We conclude that the absence of Us3 is responsible for the alterations in the Golgi architecture, while the epitope-tagged gK-HA has no obvious impact on Golgi disorganization. Unprocessed images of this, and all other figures, are available as Underlying data (Tobler et al., 2019).\n\nThe inner nuclear membrane (INM) (i) formed invaginations and multiple folds shown in detail in the inset. Capsids (c) bud at the INM, and virions accumulate in the perinuclear space delineated by the INM and outer nuclear membrane (ONM) (o), and to a much less extent in adjacent cisternae of the endoplasmic reticulum (RER). A few naked capsids (nc) are in close vicinity to Golgi membranes (Go). Note the presence of electron dense material (p) with the appearance of proteins in the nucleus and the cytoplasm. Bars, 200 nm.\n\n(A) homogenous, proteinaceous deposits (p) adjacent to altered Golgi membranes (Go), and (B) Golgi fields containing thick, electron dense membranes with the staining properties of proteins. Note the virion within double-coated structures (arrow), and the one in the endoplasmic reticulum (beside the arrowhead). Bars, 200 nm.\n\nTransmission electron microscopy of Vero cells 24 hpi (A) and 20 hpi (B) with CK177ΔUs3gK-HA. The nucleus in (A) is tangentially sectioned so that invaginations of the inner nuclear membrane (arrow) appear as hibernations. The Golgi complex (Go) comprises multiple stacks consisting of electron dense membranes similar as in R7041ΔUs3 infections. One capsid (c) is in the cytoplasm, and two virions (one indicated by “v”) are within small concentric vacuoles. Note the well-preserved mitochondria (m). (B) shows details of Golgi membranes, which continue into rough endoplasmic reticulum membranes (thick arrow), and a tangentially sectioned Golgi field. Bars, 200 nm.\n\nBudding of capsids at nuclear and Golgi membranes starts by insertion of budding proteins that appears in electron micrographs as a dense layer (Leuzinger et al., 2005). The major budding proteins at nuclear membranes are UL31/UL34 (Bigalke et al., 2014; Hagen et al., 2015), while gK and UL20 protein are responsible for budding at Golgi membranes (Melancon et al., 2007). Infection with CK177gK-HA revealed that gK localizes in cytoplasm, nuclear rim and even in nuclei (Figure 4A) by immunofluorescence using antibodies against HA. Following infection with CK177ΔUs3gK-HA, gK-HA signals are strongly enhanced in the cytoplasm (forming large clusters), at the nuclear rim and in nuclei (Figure 4B). At the ultrastructural level, Golgi membranes (Figure 5), nuclear membranes and viral envelopes (Figure 6) and the homogeneous structures in nuclei and cytoplasm (Figure 7) were distinctly labeled in CK177ΔUs3gK-HA infected cells. Immunogold labeling agrees with the distribution of gK-HA visualized by immunofluorescence implying that gK was, in addition to Golgi localization, translocated into nuclei, incorporated into nuclear membranes, and became part of the viral envelope during budding.\n\nImmunofluorescence microscopy of Vero cell at 20 hpi with CK177gK-HA (A) or CK177ΔUs3gK-HA (B). CK177gK-HA infected cells show fine but dense distribution of gK-HA over the entire cytoplasm, at the nuclear rim and, at a less extent, in the nucleus. Distribution of Us3 is similar though more intense at the nuclear rim. In the absence of Us3, gK-HA signals are markedly enhanced in all three compartments. gK-HA partly localizes with wheat germ agglutinin (WGA) as a marker for Golgi membranes. The fate of the Golgi complex is under investigation using specific markers.\n\nGold particles are irregularly distributed on electron dense Golgi membranes (Go) as well as on proteinaceous deposits (p). Bars, 200 nm.\n\n(A) Shows an overview. (B) Shows a detail of membrane folds protruding into the nucleus. Gold particles are located on nuclear membrane folds (f), virions (v), nuclear membranes (i, o) and on protein deposits (p) in the nucleus. Bars, 200 nm.\n\nAfter infection with CK177ΔUs3gK-HA, homogenous structures are intensely labeled by antibodies against HA in nuclei (A) and cytoplasm where they often neighboring Golgi membranes (Go). Bars, 200 nm.\n\n\nDiscussion\n\nThe precise functions of Us3 kinase has been reviewed by Kato & Kawaguchi (2018). However, the mechanism of regulation phospholipid-biosynthesis is unknown (Wild et al., 2012). Membrane enlargement in the absence of Us3 is associated with structural alterations including folding of nuclear membranes and malformation of Golgi stacks, respectively, and insertion of proteins into nuclear and Golgi membranes. Immunolabeling using antibodies against the HA tag recognized gK to be one component of altered membranes suggesting that deposition/insertion of gK is related to membrane overproduction in the absence of Us3.\n\nHSV-1ΔUs3 virions are infective even though they are not released from the PNS, and do not pass the Golgi complex. gK has been reported to be involved in viral envelopment by Golgi membranes, not by nuclear membranes (Melancon et al., 2005). gK is essential for infectivity playing a significant role in viral entry (Foster et al., 2001; Musarrat et al., 2018). Therefore, gK must be provided during budding of HSV-1ΔUs3 at nuclear membranes. The intense labeling of gK on nuclear membranes and viral envelopes clearly demonstrates that gK becomes part of the viral envelope during budding of HSV-1ΔUs3. gK was also found in nuclei in both HSV-1ΔUs3 and CK177gK-HA infected cells indicating that gK is transported into the nucleus in the presence or absence of Us3. Indeed, others have also observed gK incorporated into nuclear membranes in the context of wtHSV-1 infection (Rajcani & Kudelova, 1998) suggesting that gK may also play a significant role in envelopment of wtHSV-1 at nuclear membranes.\n\n\nConclusion\n\nWithout Us3, Golgi and nuclear membranes proliferate in association with incorporation of gK that also localizes on virions remaining stuck in the PNS but mature to infectivity suggesting that i) Us3 kinase is involved in regulation in nuclear and Golgi membranes assembly possibly in association with gK synthesis and/or distribution, ii) gK may be the target of Us3 phosphorylation, which is needed for virions to proceed out of the PNS, and iii) gK may also be involved in nucleus-to-cytoplasm translocation in wtHS-1 because gK localizes on nuclei and nuclear rim in the presence of Us3. We hypothesize that Us3 interplays with mechanisms regulating synthesis and arrangement of Golgi membranes, nuclear membranes and gK, which might be the most significant role of Us3 remaining to be investigated.\n\n\nData availability\n\nFigshare: The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K. https://doi.org/10.6084/m9.figshare.8131241.v2 (Tobler et al., 2019).\n\nThis project contains the raw images captured for each Figure, with the number of the Figure indicated on each file name.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis study was supported by the Foundation for Scientific Research at the University of Zürich, Switzerland.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Bernard Roizman, University of Chicago, for R7041ΔUs3 and antibodies against Us3.\n\n\nReferences\n\nBaines JD, Ward PL, Campadelli-Fiume G, et al.: The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress. 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Kousoulas", "expertise": [ "Reviewer Expertise Molecular virology and immunopathogenesis of herpes viruses" ], "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 results are presented indicating that deletion of the HSV-1 US3 causes internal membrane proliferation and rearrangements and is accompanied by over expression of glycoprotein K (gK). The results point into a potential functional relationship between US3 and gK that has not been previously reported. Authors should note that a previous publication showed that overproduction of gK causes accumulation of enveloped capsids inside the nuclear membranes. This reference should be included and discussed. Authors should also sequence the gK region to ensure that the overproduction of gK is not independent of US3 due to secondary mutations in the genomic area.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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": "49006", "date": "08 Jul 2019", "name": "Antonio Alcamí", "expertise": [ "Reviewer Expertise molecular virology and viral pathogenesis", "herpesviruses and poxviruses." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHSV-1 accumulates in the perinuclear space when US3 is deleted. The authors investigate the distribution of gK in the context of US3 deletion. gK is a glycoprotein proposed to be involved in the envelopment of capsids by Golgi membranes, but not by nuclear membranes. The incorporation of gK into the virus envelope in spite of virus morphogenesis being arrested in the perinuclear space in the absence of US3 suggests new functions of gK during budding of HSV-1 at the nuclear membranes. This is a novel observation in relation to the function of gK in viral morphogenesis.\n\nThe authors generated two new recombinant viruses, CK177deltaUS3gK-HA and CK177gK-HA, using the BAC system. A major concern of the experimental work presented is the limited characterization of the genomic structure of these recombinant viruses. The possibility that undesired mutations in other loci of the viral genome may have been selected during the construction of the recombinant viruses should be controlled by either sequencing of the viral genome or the construction of a revertant virus in which the modification (introduction of gK-HA with or without the deletion of US3) is restored, and demonstration that the revertant viruses behave as WT viruses. Also, there is no evidence that US3 protein expression is absent in CK177deltaUS3gK-HA. A detailed characterization of the recombinant viruses is necessary to fully support the conclusions of the study, and to rule out the introduction of mutations in other genes.\n\nThe presentation of more quantitative data to support the distribution of gK by immunoelectron microscopy would strengthen the conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? 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? No", "responses": [] }, { "id": "51383", "date": "02 Aug 2019", "name": "Benedikt Kaufer", "expertise": [ "Reviewer Expertise Herpesvirus replication and pathogenesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors generated two herpes simplex virus 1 (HSV-1) mutants harboring a deletion of US3 and insertion of a fluorescent tag on gK. They used these viruses to assess the effect of the US3 deletion on the gK expression levels and localization. Intriguingly, they observed an increased expression of gK and localization of the protein to the nuclear lamina. The manuscript is exciting and well written. However, as the other reviewers pointed out, it would be worth sequencing the recombinant BACs (or the resulting viruses) to ensure that the effect is not due to secondary mutations in the virus genome. Nowadays this would be quick and relatively cheap to sequence the virus genome by miSeq and would eliminate the need for a revertant virus.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-727
https://f1000research.com/articles/8-719/v1	23 May 19	{ "type": "Study Protocol", "title": "A phase IIb study to determine the safety and efficacy of candidate INfluenza Vaccine MVA-NP+M1 in combination with licensed InaCTivated inflUenza vaccine in adultS aged 65 years and above (INVICTUS): a study protocol", "authors": [ "Hannah Swayze", "Julie Allen", "Pedro Folegatti", "Ly-Mee Yu", "Sarah Gilbert", "Adrian Hill", "Chris Ellis", "Christopher C. Butler", "Julie Allen", "Pedro Folegatti", "Ly-Mee Yu", "Sarah Gilbert", "Adrian Hill", "Chris Ellis", "Christopher C. Butler" ], "abstract": "Seasonal influenza has a significant annual global impact. Current influenza vaccines work by inducing strain-specific antibodies against the highly polymorphic surface proteins of the influenza virus and need to be redesigned every year, increasing their cost and limiting availability. There is a demand for a more efficacious vaccine, particularly in older adults in which the current vaccines show poor efficacy. The aim is to investigate a novel vaccine, MVA-NP+M1, which targets T cell responses to the nucleoprotein and matrix 1 core proteins of the influenza virus A, which are highly conserved, and therefore may provide long protection against a broad range of influenza strains. INVICTUS is a phase IIb study to determine the safety and efficacy of candidate INfluenza Vaccine MVA-NP+M1 in combination with licensed InaCTivated inflUenza vaccine in adultS aged 65 years and above is a randomised, participant-blinded, placebo-controlled, multi-centre phase IIb efficacy study planned for 2030 volunteers aged 65 and over, in primary care. The primary objective is to assess the efficacy of MVA-NP+M1 co-administered with licensed inactivated quadrivalent influenza vaccine in adults ≥65 years. Participants complete daily diaries to record solicited and unsolicited events in the first four weeks post vaccination, and influenza-like illness (ILI) symptoms and severity throughout the influenza season. We hypothesise an improvement in the primary outcome, a reduction in the average number of days spent with moderate or severe influenza-like illness during periods of influenza circulation, in the group administered with MVA-NP+M1, compared to those in the control group. Registration: ClinicalTrials.gov identifier NCT03300362. Protocol version: INVICTUS Protocol v3.0, 08 June06 2018.", "keywords": [ "Influenza", "vaccination", "primary care", "infection", "MVA-NP+M1", "older adults" ], "content": "Abbreviations\n\nAE, adverse event; CI, chief investigator; CRF, case report form; DMEC, data monitoring ethics committee; GCP, good clinical practice; GP, general practitioner; ICH, International Conference on Harmonisation; MHRA, Medicines and Healthcare products Regulatory Agency; MVA, modified vaccinia virus Ankara; NP, nucleoprotein; Pfu, plaque forming units; PIS, participant information sheet; QA, quality assurance; REC, research ethics committee; SAE, serious adverse event; SMPC, summary of medicinal product characteristics; SUSAR, suspected unexpected serious adverse reactions.\n\n\nIntroduction\n\nSeasonal influenza has a significant annual global impact accounting for an estimated 1 billion illnesses and 250,000–500,000 deaths1 with an estimated economic cost of $87.1 billion in the US alone2. The unpredictable risk of sporadic outbreaks of human infections with avian influenza (H5N1) could trigger a new pandemic if the virus acquires the ability to transmit from person to person3.\n\nVaccination remains the most cost-effective strategy available to combat influenza. Current influenza vaccines work by inducing strain-specific antibodies against the highly polymorphic surface proteins (haemagglutinin, neuraminidase) of the influenza virus. Vaccines are produced based on a prediction of strains likely to circulate in the population in the upcoming influenza season. The need for constant redesign and remanufacture increases cost and places limitations on vaccine supply4, potentially leaving large populations susceptible to infection and illness. Efficacy of currently available influenza vaccines is significantly reduced in older adults (30–40%) compared to younger groups (70–90%)5, which highlights the need for better protection in this age group.\n\nWhen individuals are exposed to new influenza virus strains, to which they lack protective neutralising antibodies, cross-reactive T-cells against conserved internal antigens of influenza are associated with less viral shedding, reduced symptoms duration and severity6,7. We constructed MVA-NP+M1, a recombinant, replication-deficient modified vaccinia Ankara (MVA) vector expressing the conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1) as a fusion protein8, in the novel immortalised duck retinal cell line AGE1.CR.pIX. The aim of the vaccine is to boost cross-reactive T-cell responses to protective levels, providing immunity to not only human seasonal influenza, but also other influenza A subtypes currently found in avian species or swine.\n\nPrior to INVICTUS, MVA-NP+M1 has been administered to 151 adults in seven clinical trials (see Table 1). The vaccine generally boosted T-cell responses when administered to healthy adults. In a small, un-randomised Phase IIa challenge study, fewer vaccinated volunteers developed influenza than unvaccinated ones and there was a trend toward a reduction in duration of virus shedding in vaccinated volunteers9. The vaccine has been shown to have a good safety profile with no vaccine related serious adverse events (SAE) in these trials.\n\n\nProtocol\n\nEthics and dissemination. The trial is conducted according to the principles of the Declaration of Helsinki, relevant regulations and good clinical practice, and will be disseminated through publication in peer-reviewed scientific journals. The study was approved by the South Central, Berkshire Research Ethics Committee (17/SC/0300).\n\nThe primary objective is to assess the efficacy of MVA-NP+M1 in combination with licensed inactivated influenza vaccine (IIV) in adults ≥65 years. See Table 3 for details of all objectives.\n\nThe trial is an individually randomised, participant-blinded, placebo-controlled, multi-centre phase IIb efficacy study in 2030 volunteers aged 65 and over, in England. The main study is set in primary care and a separate immunology sub-cohort of 100 participants will be recruited at the Jenner Institute, University of Oxford. The trial will be promoted through various media sources.\n\nParticipants are randomly assigned to either the control (licensed IIV and placebo) or intervention group (licensed IIV and MVA-NP+M1) by the Research Nurse.\n\nMVA-NP+M1. The vaccine has been described previously and consists of a replication deficient MVA viral vector expressing the NP and M1 antigens from the influenza A virus (H3N2, 87 A/Panama/2007/99) as a single fusion protein8. A dose of 1.5 ×108 pfu will be used. MVA-NP+M1 is manufactured under good manufacturing practice conditions by Emergent Biosolutions, USA, and is certified and labelled for trial at the Clinical Biomanufacturing Facility, University of Oxford. The vaccine is stored at -80°C in a temperature monitored, secure freezer at the clinical sites.\n\nSeasonal influenza vaccine (licensed IIV). Pre-packaged needle and syringe containing 0.5 ml licensed IIV (Split Virion) is stored at 2°C to 8°C in a secure, temperature-monitored refrigerator at site.\n\nPlacebo. Participants allocated to the control group will receive an intramuscular injection of 0.9% saline. The volume and site of injection will be the same as for the MVA-NP+M1 group.\n\nInformed consent. Once eligibility has been confirmed (Table 2), participants provide written informed consent before any study procedures are performed. The participant is fully informed of all aspects of the trial, the potential risks and their obligations, and will be able to ask questions.\n\nVaccination procedure. The seasonal influenza vaccine is administered first, and then saline or MVA-NP+M1 administered no longer than 5 minutes after, within a 1cm circle. All vaccines are administered intramuscularly into the deltoid region of the arm.\n\nParticipant observation. Participants are observed for a minimum of 10 minutes after vaccination. Medicines and resuscitation equipment are available for the management of anaphylaxis. Participants are provided with an oral thermometer, follow-up diaries and a card with study contact details. An out of hours telephone number is provided.\n\nVaccination postponement. Vaccination may be postponed in any of the following events at the time of vaccination.\n\nAcute disease\n\nTemperature of >37.5°C.\n\nReceipt of a licensed inactivated vaccine within 2 weeks prior to enrolment.\n\nReceipt of a licensed live vaccine within 4 weeks prior to enrolment.\n\nA sub-cohort of participants (50 for each of two seasons) will be recruited to assess the immunogenicity of MVA-NP+M1 in combination with the licensed IIV. In addition to the procedures described for the main cohort, the sub-cohort attend a screening visit, and three further visits where blood samples are taken for monitoring of laboratory AEs and immunology purposes at weeks 1, 3 and 26. Blood samples will be stored in compliance with the Human Tissue Act (2004).\n\nParticipant Diaries. Participants record their symptoms in electronic diaries or paper diaries. Daily or weekly reminders are sent.\n\nWeek 1 diary. Participants are asked to record the occurrence and severity of solicited and unsolicited adverse events for one week. The following solicited adverse events are recorded by participants: local (pain, redness, warmth and pruritus at injection site) and systemic (feverishness, chills, myalgia, fatigue, headache, nausea, vomiting, arthralgia, malaise and oral temperature).\n\nWeek 2–4 diary. From weeks 2–4, participants report unsolicited adverse events, and influenza-like illness (ILI): feverishness, cough, sore throat, malaise, headache, myalgia, shortness of breath, runny/blocked nose, sneezing and temperature. For the duration of ILI symptoms they record: the severity of symptoms (mild, moderate or severe), oral temperature and any new medication.\n\nDaily and symptom diary. From week 5 until the end of the flu season, participants are only required to report ILI as described for weeks 2–4.\n\nSafety follow-up. Participants are contacted at 24 (+48 h) hours, on day 7 (+2 days) and every 3–4 weeks after vaccination, to monitor and record the occurrence of any SAEs and GP consultations, remind participants to complete their diary and to collect ILI data when a diary hasn’t been completed.\n\nA medical notes review is conducted to collect details of any occurrences in the follow-up period of: laboratory-diagnosed influenza illness, medically attended respiratory illness, GP consultations, antibiotic prescriptions and hospitalisations.\n\nDetails can be found in the Data Management Plan, held at the PC-CTU, University of Oxford. The plan is also available as Extended data10.\n\nConfidentiality. Participants will be identified only by a participant ID number. The trial will comply with the Data Protection Act 2018. All documents will be stored securely and only accessible by trial staff and authorised personnel, at the University of Oxford.\n\nAccess to data. Vaccitech Ltd. and the University of Oxford will have access to the final trial dataset. GP sites will have access to the dataset for participants registered at their practice. Access to the data will be outlined in the relevant contract.\n\nPrimary endpoint. The primary endpoint is the number of days with moderate or severe ILI during the influenza season defined using UK surveillance data. A protocol ILI definition will be adopted for the purposes of analysis: feeling feverish or having a fever (temperature ≥37.8°C) and at least one of the following symptoms: a cough and/or sore throat. See Table 3. for details of all primary, secondary and tertiary endpoints.\n\nThe study team obtain the randomised group allocation through a validated and secured web-based server (Sortition®). Randomisation will use non-deterministic minimisation on practice, age and gender to ensure each arm is balanced and 1:1 allocation when all participants have been recruited. The immunology sub-cohort will be randomised separately using the same method. Emergency randomisation procedure is also available if the web-based system isn’t accessible.\n\nThe participant is blinded to the group they have been allocated to, but the staff administering the vaccine aren’t blinded. Investigators recording and assessing clinical and safety outcomes are blinded to group allocation.\n\nWe assumed the average number of days spent with moderate or severe ILI per “season” of circulation was 3.5 days and a typical follow-up period for a season of approximately 120 days across this corresponds to 2.92% of days [6]. A sample size of 2,030 (1,015 per group) will provide 85% power to detect a relative drop of 20% in the primary outcome between the two groups (from 2.92% to 2.34%) at 5% level of significance (2-sided). This sample size has accounted for a 25% attrition rate and a further 15% increase to account for clustering of participants within households, which are estimated to be 0.04.\n\nFor the secondary outcomes, we estimated that 12.25% of vaccinated individuals over 65 years old experience an ILI most likely attributable to influenza [6].The proposed sample size will have 90% power to a reduction in the percentage of individuals experiencing ILI from 28.5–22.25%. We estimate a 50% improvement in vaccine efficacy over the IIV alone in reducing the occurrence of ILI due to influenza during periods of influenza circulation.\n\nDescription of statistical methods. The primary statistical analysis will be by intention-to-treat (ITT). Between group comparisons will be presented using effect measures (ratio or difference in response rate, difference in means) with 95% confidence intervals.\n\nThe primary outcome will be modelled using a generalised linear mixed effect logistic model, adjusting for flu season (first or second season), age, sex, GP practice and chronic illnesses. The outcome will be presence of any of the listed symptoms (feverishness, cough, sore throat, malaise, headache, myalgia, shortness of breath and temperature) rated as moderate or severe by participants who meet the protocol ILI definition on any of the days that are included for study purposes. Random effects will be incorporated to account for the clustering of participants within households and to account for the clustering of data over days within participant.\n\nComparison between groups of the proportion of participants experiencing an ILI will be carried out using the same method as the. The mixed effects will acknowledge the clustering of participants within households. The effect measure presented for MVA-NP+M1 compared to IIV alone will be an adjusted odds ratio with 95% confidence interval, which can be interpreted as an adjusted relative risk given the low prevalence.\n\nWe will use mixed-effect model, or an equivalent nonparametric method where appropriate, to analyse continuous secondary outcomes, adjusting for the same baseline measures and stratification variables. We will compare the safety outcomes using Chi-squared or Fisher’s exact test.\n\nAll the tests will be done at a 5% two-sided significance level. Data analysis will be restricted to the periods where acute respiratory illnesses are likely to be attributable to influenza based on local and national surveillance intelligence.\n\nImmunogenicity analyses. In the immunology sub-cohort, the primary analysis will be to assess the difference in magnitude of influenza-specific T-cell (using interferon gamma enzyme-linked immunospot assay) and antibody responses (using influenza hemagglutination inhibition assay) between the two groups. We will assess the vaccine immunogenicity by comparing the change in these immunological parameters from baseline to 21 days after vaccination between the groups, and how this varies according to baseline factors of age, gender and chronic illness. To assess the durability of immune responses post-vaccination, the stability of immunological parameters between the post vaccine time point and end of season time point will be assessed. The immunological x will be log transformed (based 10) and we will use appropriate regression or correlation methods to carry out the analysis.\n\nReporting procedures for adverse events. All adverse events (AEs) occurring in the first 28 days post-vaccination are captured in the diaries. The relationship of each AE to the trial medication is determined by a medically qualified individual. All AEs that result in a patient’s withdrawal from the study will be followed up until a satisfactory resolution occurs, or a non-study related causality is assigned. All reported severe symptoms are reviewed by the Medical Monitor in the first 4 weeks.\n\nReporting procedures for SAEs. All SAEs are documented and reported according to the Pharmacovigilance SOP. The assessment of expectedness is made by a clinician using the reference safety information current at the time of the event. If deemed necessary un-blinding will occur in accordance with trial specific working instructions.\n\nGroup holding rules. If a group holding rule is activated, further vaccinations won’t occur until it is deemed appropriate to restart, by the MHRA: if more than 30% of vaccinations are followed by the same Grade 3, solicited local AE or solicited systemic AE or unsolicited AE, beginning within 2 days after vaccination and persisting at Grade 3 for >48 hrs. Other events that trigger the rule include related death, life-threatening reaction, SUSAR, acute allergic reaction or anaphylactic shock. The study can also be put on hold upon advice of the Chief Investigator, Sponsor, MHRA, REC or DMEC.\n\nParticipants may withdraw voluntarily, on the decision of the Investigator or on the advice of the DMEC. If a participant withdraws from the study, data and blood samples collected before their withdrawal will be used/stored unless they specifically requests otherwise.\n\nRegular monitoring at investigator sites is performed according to ICH GCP and a monitoring plan.\n\nThis study is sponsored and funded by Vaccitech Ltd.\n\nA joint DMEC and Trial Steering Committee periodically reviews and evaluates the accumulated study data for participant safety, study conduct and progress, and efficacy. The committee includes three appropriately qualified members. The DMEC will review SAEs deemed at least possibly related to study interventions.\n\nThree interim safety reviews are planned to evaluate the safety data accumulated up to the following time points (by the DMEC):\n\n1. After the first 100 participants have been randomised\n\n2. The end of vaccinations in the first year of the study\n\n3. The end of the 1st year of follow-up\n\n\nDiscussion\n\nThere is an urgent and pressing demand for improved vaccination strategies against a broad spectrum of influenza virus strains. INVICTUS aims to investigate whether MVA-NP+M1 is capable of providing protection against a broad spectrum of influenza A virus strains with better immune responses in older people, who are at higher risk of severe influenza disease.\n\nINVICTUS is the first clinical trial to investigate whether the combination of MVA-NP+M1 and the seasonal vaccine, reduces ILI symptoms in patients, which would be both clinically useful and considerable step forward in vaccine development.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nOpen Science Framework. F1000Research INVICTUS Protocol Manuscript. https://doi.org/10.17605/OSF.IO/T6P2B10.\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nSPIRIT checklist for “A phase IIb study to determine the safety and efficacy of candidate INfluenza Vaccine MVA-NP+M1 in combination with licensed InaCTivated inflUenza vaccine in adultS aged 65 years and above (INVICTUS): a study protocol”. https://doi.org/10.17605/OSF.IO/T6P2B10.", "appendix": "Grant information\n\nVaccitech Ltd. funded the trial. The authors declare that no other grants were involved in supporting this work.\n\n\nReferences\n\nWHO: Influenza. fact sheet No. 211. 2014. Reference Source\n\nMolinari NA, Ortega-Sanchez IR, Messonnier ML, et al.: The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine. 2007; 25(27): 5086–96. PubMed Abstract \| Publisher Full Text\n\nBabakir-Mina M, Balestra E, Perno CF, et al.: Influenza virus A (H5N1): a pandemic risk? New Microbiol. 2007; 30(2): 65–78. PubMed Abstract\n\nBardenheier BH, Strikas R, Kempe A, et al.: Influenza vaccine supply, 2005-2006: did we come up short? BMC Health Serv Res. 2007; 7: 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHannoun C, Megas F, Piercy J: Immunogenicity and protective efficacy of influenza vaccination. Virus Res. 2004; 103(1–2): 133–8. PubMed Abstract \| Publisher Full Text\n\nHayward AC, Wang L, Goonetilleke N, et al.: Natural T Cell-mediated Protection against Seasonal and Pandemic Influenza. Results of the Flu Watch Cohort Study. Am J Respir Crit Care Med. 2015; 191(12): 1422–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSridhar S, Begom S, Bermingham A, et al.: Cellular immune correlates of protection against symptomatic pandemic influenza. Nat Med. 2013; 19(10): 1305–12. PubMed Abstract \| Publisher Full Text\n\nBerthoud TK, Hamill M, Lillie PJ, et al.: Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin Infect Dis. 2011; 52(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLillie PJ, Berthoud TK, Powell TJ, et al.: Preliminary assessment of the efficacy of a T-cell-based influenza vaccine, MVA-NP+M1, in humans. Clin Infect Dis. 2012; 55(1): 19–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwayze H: F1000Research INVICTUS Protocol Manuscript. 2019. http://www.doi.org/10.17605/OSF.IO/T6P2B" }	[ { "id": "53395", "date": "24 Sep 2019", "name": "Tamar Ben-Yedidia", "expertise": [ "Reviewer Expertise Immunology", "influenza vaccines" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes a Ph2b clinical trial to evaluate the safety and efficacy of a universal vaccine candidate against influenza. The novel vaccine is a fusion protein of MVA, with NP and M1 viral proteins that are conserved and hence, are expected to provide broad cross strain protection. The vaccine candidate activates cell mediated immunity. The trial focuses on the at-risk elderly group that will be administered once with the experimental vaccine together with the licensed inactivated vaccine against flu. A sub cohort will be tested for immunogenicity.\nMain study: There are multiple viruses that cause ILI (other than Influenza) that are not expected to be affected by this vaccine. It is not clear why the sponsor expects to have reduced influenza like illness by immunizing against Influenza. Why they do not confirm Influenza illness (e.g. by PCR) and evaluate the reduction of illness in this sub population.\nSub cohort: The specificity of the immunological parameters is not described. Do they plan to evaluate the response to the vaccine Ags? or to different influenza viruses/antigens? How will they show the broad response to the vaccine?\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "54948", "date": "24 Jan 2020", "name": "Xavier Saelens", "expertise": [ "Reviewer Expertise Influenza vaccine development", "influenza 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 manuscript describes the study protocol of a planned phase IIb study (INVICTUS) that aims to determine the safety and efficacy of an MVA vectored influenza NP-M1 fusion construct combined with a conventional influenza vaccine in adults aged 65 years or older. The conventional influenza vaccine (split virion) will be administered by standard intramuscular injection followed, within 5 minutes, by saline (placebo) or MVA-NP + M1 vaccine, also injected intramuscularly close to the influenza vaccine injection site. The trial intends to recruit 2030 volunteers, 1:1 randomized over the 2 study arms. The study will for the most be conducted in primary care settings in the UK. An immunology sub-cohort of 100 volunteers is included as well for the monitoring of seroconversion (influenza hemagglutination inhibition) and cellular responses (ELISPOT). The primary outcome is to assess the efficacy of MVA-NP + M1 combined with a conventional inactivated influenza vaccine in terms of the number of days the volunteers experience moderate or severe influenza-like symptoms based on a self-reported symptoms. Secondary outcomes include comparison of the occurrence of hospitalization and death due to respiratory illness, the rate of laboratory confirmed influenza (based on RT-PCR) and the magnitude of the immune responses.\nIn a WHO bulletin published in November 2017, it is recommended to omit “sore throat” from the clinical case definition of influenza-like illness. This may be taken in consideration for the primary endpoint determination.\nThe peptides that will be used to determine the magnitude of the influenza-specific T cell response should be specified. Only NP and M1-derived peptides? Corresponding to the Panama/99 virus or also to recently circulating influenza viruses? The analysis of the breadth of the antibody responses could be better defined.\nThe statement on influenza vaccine efficacy in older adults should be supported by more recent references (e.g. Beyer et al. Vaccine, 20131; Demicheli et al., Cochrane DSR, 20182).\nThe first sentence of the 3rd paragraph under Statistics is incomplete.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "51322", "date": "11 Feb 2020", "name": "Rebecca J. Cox", "expertise": [ "Reviewer Expertise Influenza Vaccine trials", "influenza immunological assays" ], "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 report is a study protocol for the INVICTUS clinical trial which is a phase IIb study to evaluate the safety and efficacy of seasonal IIV in combination with MVA_NP+M1. The protocol is well written and comprehensive.\n\nThere are a couple of minor issues which need to be addressed:\nAbstract: long protection should perhaps be changed to long term protection.\nPage 5: the geometric titres of neutralising antibodies are listed as outcome measures, this should be changed to HAI titres as the protocol only specifies that HAI tests will be conducted. If neutralising titres are also measured by MN or vN assays this can remain.\nPage 7: this sentence is incomplete; comparison between groups of the proportion of participants experiencing an ILI will be carried out using the same method as the.\nPage 7: it is not entirely clear what immunological x is; the immunological x will be log transformed (based 10) and we will use appropriate regression or correlation methods to carry out the analysis.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/8-719
https://f1000research.com/articles/8-705/v1	21 May 19	{ "type": "Research Article", "title": "Exploring the optimal duration of video recording in a post-discharge eLearning platform for cardiac patients", "authors": [ "Borut Kirn" ], "abstract": "Background: On a post-discharge eLearning platform for patients with coronary artery disease, videos are used as the main educational media. Medical content takes a long time to be thoroughly explained, frequently exceeding the viewer’s attention span. To find the optimal duration for such an educational video, we studied the retention of video watching. Methods: In this study, 135 (88% male; age 62±9 years) patients with coronary artery disease actively used eLearning platform which included 60 video recordings with duration ranging from 21 sec to 303 sec. The videos were divided into two groups based on their duration (short < 100 sec and long > 100 sec). From the platform usage metadata, an average video retention rate was obtained as a ratio of video viewing time and total video duration for videos watched six times or more. Independent t-tests for mean values and f-test for variance were used to compare the groups. Results: In total, 35 (18 short and 17 long) videos were included in the study and were viewed (mean ± SD) 22 ± 11 times. The mean duration of short videos was: 80 sec ± 14 sec and of long videos it was: 160 sec ± 51 sec. The retention rate in the short and long group was 0.99 ± 0.05 and 0.94 ± 0.19 respectively. Average values were not significantly different (P = 0.33), but variances were (P < 0.05). Conclusions: This study shows that the effective attention span for a video recording of this kind, based on an eLearning set-up and local population, is up to 100 sec. Videos shorter than 100sec are mostly watched fully without a break. In contrast, longer videos are often rewound, and parts watched again or not watched to the full length at all.", "keywords": [ "e-Learning", "cardiac rehabilitation", "coronary disease", "video", "tele-medicine" ], "content": "Introduction\n\nOne issue surrounding recovery at home is poor utilization of instructions given to patients after being discharged from a clinic. It has been shown that patients and their families often experience information overload while at the clinic and often forget or misinterpret up to 85% of the instructions received1,2.\n\nTo solve this problem, eLearning has been put forward as a novel mode of delivering instructions to patients3–6. In eLearning, reliable medically and scientifically sound videos are used to educate patients and their informal caregivers about the etiology, pathophysiology and treatment of their medical condition, doing so by addressing the following questions: “What has happened?”, “What can you do by yourself?” and “Who can help you?”. Video “lectures” given by doctors, nurses, physiotherapists, psychologists and dietitians are recorded and edited into topic-related modules. In addition, peer patients share best recovery practice and experiences. Selected videos are complemented by simple texts, a short quiz or illustrations.\n\nMost educational information is conveyed to the patients and their informal caregivers in video format. Therefore, it is crucial that the design of these recordings is thought through and executed carefully. The video recordings are obtained in raw format from interviews and are then post-processed to concisely deliver the required medical content. Deciding the duration of the video recording presents a challenge. If a video is long enough to fully explain the complex medical condition, there is a risk that the patient skips the video or stops watching midway, given that the usage of eLearning platforms is based solely on personal motivation. The optimal video duration to fulfil both requirements, to fully convey the medical content while retaining viewer attention, however, is not known. The purpose of this study was to investigate the impact of video duration on cardiac patients’ viewing behavior.\n\n\nMethods\n\nWithin the scope of a large randomized clinical study7 conducted in Jessa Hospital, Hasselt, Belgium and in collaboration with Primarius platform developers ClinicalTrials.gov registration number NCT02475967. Ethical approval was given by the Ethical Committee of Jessa Hospital, Hasselt, Belgium (approval number 15.82/CARDIO15.11) and all included patients provided written informed consent. The metadata provided by Viidea video streaming provider were analyzed. The analysis included 135 patients with coronary artery disease (88% male; age, 62±9 years) which actively used the provided post-discharge eLearning platform. Upon discharge, patients received a one-month access to a secure eLearning platform via a voucher containing anonymous personal login codes with which the contents were accessed, this way also assuring anonymity of the patients with respect to metadata acquisition. The patients used the eLearning platform at home and without supervision.\n\nThe content of the platform was composed of 20 topics, containing information about what has happened, what they can do by themselves and who can help them, presented from three perspectives—those of the clinician, patient and informal caregiver. Each topic contained three videos. In total there were 60 video recordings with duration ranging from 25 to 300 sec (Figure 1).\n\nThe videos were divided into two groups based on their duration. The short group included videos with duration <100 sec and the long group included videos with duration >100 sec. The video retention rate was defined as the ratio between video viewing time and total video duration. In addition, videos were divided into group featuring doctors and videos featuring peer-patients. Only videos that were watched six times or more were included in the analysis.\n\nIndependent t-tests for mean values and f-test for variance were used (Microsoft Excel 2016) to compare retention rates between the short videos and long videos groups. The differences in retention rate between the videos featuring doctors and videos featuring peer-patients were also determined.\n\n\nResults\n\nOut of 60 available videos 35 were watched six times or more and were thus included in the analysis. They were viewed on average 22 ± 11 times (Figure 2). Of the analyzed videos, 22 featured peer-patients and the rest (13 videos) featured (para-) medical professionals. With respect to their duration, 18 videos were classified as short, with an average duration of 80 sec ± 14 sec, and 17 were classified as long, with an average duration of 160 sec ± 51 sec (Figure 2). Individual results for each video are available as Underlying data8.\n\nThe videos with duration from 54 sec to those up to and including 99 sec were part of the short video group and videos with duration of 100 sec to 303 sec were part of the long videos group. The short videos group was composed of 12 videos featuring peer-patients and 6 videos featuring (para-) medical professionals. They were viewed from 8 to 56 times.\n\nIn Figure 3 the retention rate of video watching is shown for each video with respect to its duration. A clear difference in the spread of retention rate is visible around the video duration length of 100 sec. The videos shorter than 100 sec have retention rate around 100%, ranging from 107% for shorter videos (60 sec) and decreasing towards 88% as videos duration increases towards 100 sec. The videos longer than 100 sec have a wide range of retention rates, ranging from 60% to 133% without any clear trend. The average retention rate of the short and long videos group was 0.99 ± 0.05 and 0.94 ± 0.19, respectively. Mean values were not significantly different (P = 0.33), but variances were significantly different (P < 0.05).\n\nNote that retention rate for videos that are shorter than 100 sec is around 100% and slowly decreases as duration is increased up to and over 100 sec, while the retention rate for longer videos (duration > 100sec) shows much larger variation. Large variation is a result of premature ending of a video watching and rewinding with repeated watching.\n\nThe timeline of meta data acquired from watching 4 different types of videos is shown in Figure 4. We found no significant difference (P=0.7) when comparing the retention of watching videos featuring patients (0.97 ± 0.14) to those featuring (para-)medical caregivers (0.95 ± 0.12).\n\nMeta data acquired from watching 4 different types of videos: short featuring doctor (A), long featuring doctor (B), short featuring patient (C) and long featuring patient (D). The legend label “present” shows watching the video in one go (dark green), “watching” shows cumulative amount of watching including replaying (red), and “replaying” indicates the amount of replaying the video (light green).\n\n\nDiscussion\n\nThis study showed that videos which are shorter than 100 seconds are typically watched by patients without pause, while the videos longer than 100 seconds either stopped watching prematurely or frequently rewound and watched again. This finding is further supported by a clearly expressed negative slope of retention rate within the short video group. As expected, the longer the videos in this group the lower the retention rate. The study included 35 video recordings out 60 available to the patients which indicates that some topics are not as interesting for the patients as it was anticipated. Out of the 35 analyzed videos, two-thirds were featuring peer-patients and one-third the (para-)medical professionals. This difference shows the large interest of patients in peer-patient experiences, at least at the initial decision level, when deciding whether to watch the video or not.\n\nThe duration of the video is undoubtedly linked to the attractiveness of the content, the explanation provided by the featured interviewee and on the difficulty of the topic being discussed.\n\nThe main result of this study should be an increase in the awareness in the eLearning content creator community that videos as a main means of conveying information and educating should be carefully designed, because the natural attention span of most patients is up to 100 seconds.\n\n\nData availability\n\nFigshare: Exploring the optimal duration of video recording in a post-discharge e-learning platform for cardiac patients. https://doi.org/10.6084/m9.figshare.81158668.\n\nThis project contains a spreadsheet featuring raw data on video watching and retention in CSV format.\n\nUnderlying data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nThe videos used in this study can be accessed free of charge for research purpose on the eLearning platform for a limited duration. Please contact the author (borut.kirn@mf.uni-lj.si) to receive access codes to this material.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\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\nKessels RP: Patients' memory for medical information. J R Soc Med. 2003; 96(5): 219–222. PubMed Abstract \| Free Full Text\n\nKoelling TM, Johnson ML, Cody RJ: Discharge education improves clinical outcomes in patients with chronic heart failure. Circulation. 2005; 111(2): 179–185. PubMed Abstract \| Publisher Full Text\n\nBartlett EE: Cost-benefit analysis of patient education. Patient Educ Couns. 1995; 26(1–3): 87–91. PubMed Abstract \| Publisher Full Text\n\nHibbard JH, Greene J: What the evidence shows about patient activation: better health outcomes and care experiences; fewer data on costs. Health Aff (Millwood). 2013; 32(2): 207–214. PubMed Abstract \| Publisher Full Text\n\nFontaine G, Cossette S, Heppell S, et al.: Evaluation of a Web-Based E-Learning Platform for Brief Motivational Interviewing by Nurses in Cardiovascular Care: A Pilot Study. J Med Internet Res. 2016; 18(8): e224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu DS, Thompson DR, Lee DT: Disease management programmes for older people with heart failure: crucial characteristics which improve post-discharge outcomes. Eur Heart J. 2006; 27(5): 596–612. PubMed Abstract \| Publisher Full Text\n\nFrederix I, Vandenberk T, Janssen L, et al.: eEduHeart I: a multi-center randomized, controlled trial investigating the usage of a post-discharge eLearning platform for cardiac patients. Third European Congress on eCardiology and eHealth in 2016.\n\nKirn B: Exploring the optimal duration of video recording in a post-discharge e-learning platform for cardiac patients. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8115866.v1" }	[ { "id": "48885", "date": "21 Oct 2019", "name": "Drago Rudel", "expertise": [ "Reviewer Expertise Teehealth and telerehabilitation services" ], "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\nBackground: On a post-discharge eLearning platform for patients with coronary artery disease, videos are used as the main educational media. Medical content takes a long time to be thoroughly explained, frequently exceeding the viewer’s attention span. To find the optimal duration for such an educational video, we studied the retention of video watching.\nComment: The background clearly highlights the addressed challenge.\nMethods: In this study, 135 (88% male; age 62±9 years) patients with coronary artery disease actively used eLearning platform which included 60 video recordings with duration ranging from 21 sec to 303 sec. The videos were divided into two groups based on their duration (short < 100 sec and long > 100 sec). From the platform usage metadata, an average video retention rate was obtained as a ratio of video viewing time and total video duration for videos watched six times or more. Independent t-tests for mean values and f-test for variance were used to compare the groups.\nComment: The methodology is clearly set and appropriate for the type of research.\nResults: In total, 35 (18 short and 17 long) videos were included in the study and were viewed (mean ± SD) 22 ± 11 times. The mean duration of short videos was: 80 sec ± 14 sec and of long videos it was: 160 sec ± 51 sec. The retention rate in the short and long group was 0.99 ± 0.05 and 0.94 ± 0.19 respectively. Average values were not significantly different (P = 0.33), but variances were (P < 0.05).\nComment: The results are clear and derive from the implemented methodology.\nConclusions: This study shows that the effective attention span for a video recording of this kind, based on an eLearning set-up and local population, is up to 100 sec. Videos shorter than 100sec are mostly watched fully without a break. In contrast, longer videos are often rewound, and parts watched again or not watched to the full length at all.\nComment: The conclusion is relevant to the findings.\nGeneral comments: The purpose of this study was to investigate the impact of educational video duration on cardiac patients’ viewing behaviour. The paper is novel in addressing the optimal video recording duration used at educational eLeraning platforms. Its conclusion is simple and clear: the videos used by patients in different therapies should be less than 1 minute long in duration. It is encouraging that a scientific approach is used to assess efficiency and effectiveness of the novel ICT based tools that will be broadly used in the near future for therapeutic purposes in different areas of medicine.\nTechnical corrections needed: The first sentence in the Methods is not clear (Study background »Within the scope of a large randomized clinical study….«). Consider revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "67714", "date": "13 Aug 2020", "name": "Horesh Dor-Haim", "expertise": [ "Reviewer Expertise Cardivascular medicine", "exercise physilogy and digital health 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\nThis study explores the optimal duration of video recording in a post-discharge eLearning platform for cardiac patients. The purpose of the study is important in order to understand the attention span of patients, providing digital educational media for secondary prevention of CVD. The scope of the study and the aim is very simple and limited to test patients' retention. The videos were divided into two groups based on their duration. The short group included videos with duration <100 sec and the long group included videos with duration >100 sec. The video retention rate was defined as the ratio between video viewing time and total video duration. Results showed mean values were not significantly different in retention rate, however, the variance was higher in the long video clip group.\nThe aim and the study are clear, however, the methodology and results analysis are not as so. Increase in retention rate is the primary goal and the null hypothesis, however, it's not demonstrated in the results. Please consolidate the means to show increased retention. If variance in retention is the primary end point, please clarify it or other methods to test video retention.\n\nStatistical analysis should be reconsidered, using more than two groups with 100 sec cut off (e.g. short/intermediate/long).\n\nFig 4 is not clear. Provide more data about the procedures in the methods.\n\nThe authors' conclusion is that videos shorter than 100sec are mostly watched fully without a break. In contrast, longer videos are often rewound, and parts watched again or not watched to the full length at all. Please provide relevant data such as number of brakes and relays.\nThe study end point and conclusion, exploring the optimal duration of video would be much more solid if authors would have provided a pre/post questioner to test patients' understanding and 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? 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? No", "responses": [] } ]	1	https://f1000research.com/articles/8-705
https://f1000research.com/articles/7-98/v1	23 Jan 18	{ "type": "Method Article", "title": "A host subtraction database for virus discovery in human cell line sequencing data", "authors": [ "Jason R. Miller", "Kari A. Dilley", "Derek M. Harkins", "Timothy B. Stockwell", "Reed S. Shabman", "Granger G. Sutton", "Kari A. Dilley", "Derek M. Harkins", "Timothy B. Stockwell", "Reed S. Shabman", "Granger G. Sutton" ], "abstract": "The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses.", "keywords": [ "RNAseq", "human cell lines", "HepG2", "HuH-7", "Jurkat", "host subtraction" ], "content": "Introduction\n\nHost subtraction is the bioinformatics process of filtering reads derived from host DNA and RNA (Daly et al., 2015). Host subtraction enriches the non-host component of sequence datasets and is especially attractive for assays involving high-throughput sequencing technologies that generate short reads in high volume, where data reduction can realize cost savings. Following host subtraction, remaining reads can be mapped to references and counted, or used as queries to sequence databases, or possibly assembled to reconstruct novel transcript or genome sequences.\n\nHost subtraction resources are needed for human cell lines that are widely used for RNA virus propagation. This includes three human cell lines Jurkat, HuH-7, and HepG2 commonly used to grow viruses or to amplify viruses suspected to be present in clinical isolates. The Jurkat line, derived from human T cells, supports replication of HIV and some Herpesviruses. Jurkat cells have been described to harbor Xenotropic murine leukemia virus-related virus (XMRV), a now famous gammaretrovirus incorrectly proposed as a causative agent of human prostate cancer and chronic fatigue syndrome (Cingöz et al., 2012). The HuH-7 and HepG2 lines, both derived from liver cells, are widely used for virus research among multiple virus families. These cell lines are permissive to the growth of several RNA viruses. Huh7 cells support Hepatitis C virus (HCV) replication (Lindenbach et al., 2005), though not all Huh-7 lines are permissive to HCV (Sainz et al., 2009). Huh-7 cells are also permissive for Dengue virus (Lin et al., 2000). In addition, Vesicular Stomatitis Virus (VSV), of the Rhabdoviridae family, replicates efficiently in HepG2 and HuH-7 (Marozin et al., 2008). HepG2 cells are permissive to Influenza virus (Ollier et al., 2004) and have been shown to harbor cell-line specific chromosomal rearrangements (Wong et al., 2000). Notably, Jurkat, HuH-7, and HepG2 cell lines are all permissive to the Paramyxovirus Sendai virus (SeV). While SeV does not cause disease in humans, infection in mice results in pneumonia in mice (Faísca & Desmecht, 2007). More importantly, SeV is extensively used as a model to study virus-host interactions since it has a similar genomic organization to pathogenic viruses that include Ebola, Marburg, Hendra and Nipah Virus.\n\nAn ideal subtraction resource for any cell line would include a complete genome sequence. Less expensive alternatives are available for cell lines derived from humans. One subtraction alternative is a reference human genome supplemented with a collection of cell-line-specific sequences. To this end, we have developed a sequence subtraction database (SDB) that permits enrichment of viral sequences through the computational depletion of host sequences. We developed an SDB, named SDB1, representing three human tissue cell lines that are often used for the detection of RNA viruses that infect humans. Reducing the size of the NGS dataset by removing host background reads allows the researcher to perform subsequent analyses (e.g. de novo assemblies, read mapping, homology searches) more efficiently and with less computational requirements.\n\n\nMethods\n\nFrozen HepG2 cells were obtained from ATCC, part number ATCC HB-8065, lot # 61983117. The HepG2 cell line is derived from a liver hepatocellular carcinoma of a 15-year-old Caucasian male. Frozen HuH-7 cells were obtained from JCRB Cell Bank, part number JCRB0403, lot # 08062010. HuH-7 is described as a well-differentiated human hepato-cellular carcinoma cell line derived from the liver or gallbladder of a 57-year-old male Japanese patient, who died in 1985. Frozen Jurkat cells were obtained from ATCC, part number ATCC TIB-152, lot # 6213515. This Jurkat cell line was derived from peripheral blood of a 14-year-old boy who was diagnosed with acute T cell leukemia. Cells were maintained following the standard recommended protocol per cell line.\n\nA single genomic DNA library was prepared per cell line. Genomic DNA was isolated from the cell line using a Qiagen genomic DNA isolation kit. Bioanalyzer analysis confirmed high molecular DNA was recovered. After Blue Pippin size selection, fragments appeared to be 290bp. NextGen paired end barcoded genomic library construction was performed with a NEBNext whole genome library prep kit. NextGen Library quantification and normalization was performed by qPCR. Each library was test sequenced with one run on an Illumina MiSeq, and then sequenced with two runs of an Illumina NextSeq 500 using the Illumina High Output Kit. Reads were demultiplexed, which removed barcodes and sequencing adapters.\n\nFour RNA libraries were prepared per cell line. Cells were either mock infected or infected with SeV as previously described (Dilley et al., 2017). In each condition, libraries were either treated to deplete ribosomal RNA or total RNA was subjected to library construction. Libraries were multiplexed and sequenced using one run on an Illumina NextSeq 500 using the Illumina Mid Output Kit.\n\nDNA and RNA sequence reads were trimmed of adapter using CutAdapt 1.8.1. RNA sequence reads were also trimmed of low quality bases, adapter sequences, and resulting short reads using Trimmomatic 0.35. The DNA read sets were not filtered based on length, quality, or number of ambiguous base calls.\n\nReads were mapped with bowtie2 (Langmead & Salzberg, 2012) version 2.2.5 with stringent parameters designed to identify and remove only those read pairs with full-length reference agreement. Reads were mapped to the NCBI GRCh38.7 human genome reference sequence using bowtie2 in global alignment mode (--end-to-end) with low sensitivity (--fast) and stringent output settings (--no-unal --no-mixed --no-discordant). This mapping selects at most one mapping per pair.\n\nCell line gDNA reads that did not map to the human genome reference were assembled per cell line with CLC NGS Cell (Qiagen Bioinformatics) version 3.22.55708 using de novo assembly with parameter “-p fb ss 200 400“. Contigs of minimum length 200 were retained and named CLCs to indicate Cell Line Contigs.\n\nThe UniVec database was downloaded from the NCBI FTP site (5456 sequences and 1,049,913 bp). Other references were downloaded from GenBank. The reference human assembly was GCA_000001405.22_GRCh38.p7_genomic (3,232,546,710 bp). Mycoplasma sequences were obtained by searching GenBank for Mycoplasma nucleotide sequences of length 500 Kbp or more (263 sequences and 232,800,861 bp). The PhiX genome sequence was NC_001422.1 (5,386 bp). The Sendai RNA reference sequences were AB855653.1 and AB855654.1 (15,384 bp each).\n\nThe RNAseq subtraction step used a sensitive mapping strategy. Although it did not use a splice-aware aligner, it relied on sensitive local alignments, the selection of at most one best alignment per read, and the subtraction of both reads of a pair if either read mapped. The mapping used bowtie2 with parameters “--sensitive-local” and “--no-unal” to map read pairs to the subtraction database SDB1. The mapping step can be parallelized with one pair of input read files per job. Each job requires RAM approximately equal to twice the database size. Our runs against the 3.3 GB SDB1 reserved 8 GB RAM and 4 threads.\n\nRNAseq reads from the Hölzer et al. (Hölzer et al., 2016) experiments were kindly provided by the authors (Martin Hölzer, personal communication; NCBI SRA accession SRP128545).\n\nFor reads characterized by BLAST, BLASTN 2.2.31+ from the NCBI BLAST+ package was used to search the NCBI nt database. BLAST was run with parameters “-outfmt7 qseqid sseqid pident length mismatch gapopen evalue bitscore staxids” to capture taxon ID and “-max_target_seqs 1” to retain the top hit per query sequence.\n\n\nResults\n\nTo obtain cell line genomic sequence, each cell line was separately subjected to DNA sequencing. For each of three cultured human cell lines, one DNA sequencing library was created with a ~300 bp insert size. Each library was test sequenced on an Illumina MiSeq platform to generate small volumes of 2x300 bp read pairs; in these pairs, reads were expected to overlap. Each library was then sequenced on two runs of an Illumina NextSeq platform to generate high volumes of 2x150 bp read pairs. Reads were trimmed with CutAdapt. The result offered 105 Gbp of sequence in 346 M read pairs, or 28X average coverage of the human genome per cell line. Further details and public accessions are provided in Table S1. The reads were filtered to eliminate pairs that provide sequence that is already present within a standard human genome reference sequence. This was done by mapping the NextSeq genomic read pairs with stringent parameters. This step filtered 94% of read pairs.\n\nTo further characterize the mapped reads, the reference mapping was scanned for high-coverage and low-coverage areas. High coverage indicate sequences present in the cell line at higher copy number than their representation in the reference sequence. Coverage over 100X represents at least 3X higher representation in the cell line than the reference. With this criteria for high coverage, the number of reference bases at high coverage was 8.6 Mbp per cell line. The average high-coverage interval was 106 bp, which is shorter than the 150 bp read length, suggesting that short tandem repeats may be expanded in the cell lines. The mapping was also scanned for low-coverage areas. Low-coverage regions shorter than read pairs would indicate rearrangements within the cell line genomes. The combined span of reference bases with coverage less than 2X was 96 Mbp and the average low-coverage interval was 265 bp. Further details are provided in Table S2.\n\nTo capture sequences present in any of the three cell lines, but absent from the reference, the unmapped reads were assembled into Cell Line Contigs (CLCs). The assembly generated about 3 Mbp in 8 K CLCs for each cell line. CLC size statistics are provided in Table S3. The average CLC size was 380 bp though there were a few large CLCs from each cell line. The largest CLC from each cell line had a partial alignment to the largest from the other two lines. Analysis of the two largest contigs per cell line showed similarity to other human or primate sequences in the public databases and redundancy between cell lines (Table S6).\n\nThe subtraction database named SDB1 was constructed by concatenating FASTA representations of the CLCs, the human genome reference, the PhiX genome, the UniVec database, and a collection of Mycoplasma complete genomes. Table S4 shows the number of sequences and number of bases per data source.\n\nThe SDB process was assessed with a test designed to emulate a cell-based RNA virus detection assay. In this assay, environmental samples are analyzed to determine which RNA viruses are present, if any. To overcome the presumed low titer of viral RNA, this assay uses virus-permissive cell lines to amplify viral load. After exposure to environmental samples, the cells in culture are grown for sufficient time for viral replication. RNA is harvested, optionally depleted of rRNA, and sequenced. The rRNA depletion step is employed to enrich the non-host RNA and increase the number of non-host RNAseq reads generated. Finally, a sequence analysis step involves alignment of the RNAseq data to reference databases for taxonomic classification and quantification. Classification of host cell reads is uninformative so all computational investment in classifying them represents overhead cost. The goal of the SDB step is to reduce the overhead without loss of sensitivity. Although the taxon of the virus was known a priori , the same process could be used to detect unknown viruses in an uncharacterized sample.\n\nThis test used SDB1, our subtraction database that includes genomic DNA sequence derived from the HepG2, HuH-7, and Jurkat human cell lines. This test also used RNAseq from those cell lines. RNAseq reads were generated from multiple samples and mapped to SDB1. This test used uninfected cells and cells infected with the Sendai virus (SeV). After the growth period, cells were treated for rRNA depletion, or left untreated as a control. Twelve libraries were generated in total, representing the three cell lines under four different conditions: infected-and-depleted, infected-and-not-depleted, mock-infected-and-depleted, and mock-infected-and-not-depleted. The libraries were barcoded , multiplexed, and sequenced on a NextSeq platform to generate 2x150 bp read pairs. RNAseq read counts per library are provided in Table S1.\n\nThe sequence data were subjected to a subtraction process by mapping to SDB1. The mapping use local alignments to allow host spliced RNA sequences to map to host genomic DNA sequence. The results are shown in Table 1. The read counts after trimming ranged from 19 to 27 million per library (Table 1, column A). The rate of subtraction ranged from 89% to nearly 100% per library (Table 1, column B). The relative contribution by each type of SDB1 sequence is shown in Table S5. As expected, the human genome reference sequence subtracted the most reads. The CLCs from the human cell lines subtracted 0.19% to 1.34% of reads per library. Contrary to expectation, the cell line origin of the RNAseq was not predictive of the cell line whose CLCs would subtract the most reads. Instead, the CLCs derived from HuH-7 consistently subtracted more RNAseq reads than those from HepG2 or Jurkat. It is possible that the CLCs derived from HuH-7 capture larger portions of transcriptional units that are partially represented in the other CLCs.\n\nThree cell lines were grown with Sendai virus (SeV) infection or mock infection. Some samples were treated with Ribo-Zero (Illumina) to deplete rRNA. (A) All cDNA libraries were sequenced on the Illumina NextSeq platform to generate over 19 million RNAseq reads per sample. (B) At least 89% of reads from every sample mapped to the subtraction database named SDB1. (C) Non-SDB reads were mapped to SeV references. Expressed as a fraction of initial reads, the SeV complement was 5% to 10% in infected samples and less than 1% in the controls. (D) Expressed as a percentage of non-SDB reads, the SeV complement was 78% to 95% in infected-but-not-depleted samples. The complement was larger in the infected-and-depleted samples, and smaller in the mock-infected samples. This suggests that analysis of non-SDB reads could support the detection of known viruses in an uncharacterized sample. The apparent false positive enrichments (e.g. 24.24% SeV in HEP/none/none) can be discounted by applying a minimum requirement for 0.10% viral reads out of initial reads.\n\nNext, the RNAseq reads that did not map to SDB1 were extracted for analysis. These “non-SDB” reads were mapped to SeV genome reference sequences. As expected, the libraries with the largest SeV complement were the libraries that had been infected with Sendai virus and depleted of rRNA. When expressed as a portion of initial reads, the SeV complement was 5% to 10% for these RNAseq libraries (Table 1, column B). When expressed as a portion of the non-SDB reads, the SeV complement was 84% to 99% (Table 1, column C). This demonstrates that the SDB subtraction step left RNAseq data that was highly enriched for the viral sequence.\n\nIn all the datasets derived from infected libraries, SeV was the majority component of the non-SDB reads (Table 1, column D). This was true even for the non-depleted libraries. When expressed as a portion of initial reads, the SeV complement was 0.47% to 0.96% for non-depleted libraries. When expressed as a portion of non-SDB reads, the SeV complement was 78% to 95% for non-depleted libraries. While the non-depleted libraries did contain fewer SeV reads in absolute terms, the portions of reads identified as SeV after subtraction were almost as high as those for the depleted libraries. This suggests that the SDB subtraction can provide a viable alternative to rRNA depletion for the detection of RNA viruses.\n\nIn the datasets from cells that were mock infected, the portion of non-SDB reads mapping to SeV was non-zero (Table 1, column D). In one case, it was 24%. These results suggest that taxonomic classification by mapping is noisy and that false positive virus identifications could result. Our data suggests a threshold for avoiding false positives, namely that a virus is suspected only if the post-SDB virus read count is as least 0.10% of initial reads. Additional data and additional replicates would assist selection of appropriate thresholds for desired levels of specificity.\n\nThe viral taxon was known beforehand and mapping non-SDB reads to SeV references only confirmed a priori knowledge. In a discovery situation, the non-SDB reads could be characterized by BLASTn homology search against the NCBI nt database. With our data, SeV was the taxon with the most hits for all libraries. This result indicates that SeV could have been detected de novo by BLAST analysis. After SeV, no single taxa was represented at over 0.01% of initial reads. The secondary taxa indicated by BLAST included Newcastle disease virus (concentrated in the one library of HuH-7 with viral infection and depletion), Ebola virus, Human ORFeome Gateway entry vector, Homo sapiens, Gorilla gorilla, and others. The virus hits are possibly indicators of contamination from simultaneous projects in the lab. The primate hits could be due to the higher sensitivity of the BLAST homology search compared to mapping.\n\nThe subtraction computation consumed 1,285 cpu sec per million reads. The BLAST analysis consumed 107,146 cpu sec per million reads. The total cost of subtraction-then-blast was approximately 20% of the hypothetical cost of running BLAST on every read.\n\nThe utility of SDB1 was tested on data from an independent source. Hölzer et al. (Hölzer et al., 2016) explored host cell expression changes in HuH-7 human cells and R06E-J cells from the bat Rousettus aegyptiacus. As part of the study, cells were infected with either the Ebola virus strain Zaire, Mayinga (GenBank:NC_002549), or the Lake Victoria Marburg virus, Leiden (GenBank:JN408064.1), or a mock infection. Cells were harvested at 3, 7, or 23 hours after infection and sequenced by RNAseq.\n\nThe RNAseq reads from the HuH-7 experiments of Hölzer et al. were downloaded and trimmed; see Table 2. From every library, a majority of reads were subtracted by mapping to SDB1 (Table 2, column B). After subtraction, the non-SDB reads were mapped to reference genome sequences for Ebola (EBOV) and Marburg (MARV). The EBOV complement of the non-SDB reads was 57% to nearly 100% in EBOV-infected libraries and lower in the other libraries (Table 2, columns C–D). The MARV complement of the non-SDB reads was 39% to 75% in MARV-infected libraries and lower in the other libraries (Table 2, columns E–F). These results confirm that SDB1 can provide effective enrichment of other viruses within independent cell cultures of HuH-7.\n\nData from an independent study (Hölzer et al., 2016) are derived from RNAseq of Huh-7 cells infected with Ebola virus (EBOV), Marburg virus (MARV), or none (Mock). The data were re-analyzed here using SDB1. Subtraction enriched the EBOV complement to at least 57% in the EBOV-infected samples. Subtraction enriched the MARV complement to at least 39% in the MARV-infected samples. The apparent false positive enrichments can be discounted by applying a minimum requirement for 0.10% viral reads out of initial reads.\n\n\nDiscussion\n\nWe analyzed the human cell lines HepG2, HuH-7, and Jurkat in order to increase their utility for amplifying and detecting viruses in clinical or environmental samples. Using low-coverage short-read sequencing of genomic DNA, we generated cell line contigs (CLCs) from reads that did not map to the human genome reference (GRCh38). The CLCs should have captured genomic breakpoints in the cell lines with respect to the human reference, though they probably do not capture solitary SNPs in cell line DNA. We demonstrated utility by mapping RNAseq reads from cells infected with RNA viruses, as well as control cells, to a database called SDB1 composed of CLC, human, and contaminant sequences. We were able to subtract host sequences and enrich each dataset for the non-host complement.\n\nFor the cell lines analyzed here, the CLCs added a small amount of mapping sensitivity to that of the high-quality GRCh38 human genome reference. CLCs from other cell lines could add more power if the cell line differs more substantially from the reference. CLCs could be constructed for experiments with cells suspected of harboring transfection-induced chromosomal rearrangements, as was reported for transfected HepG2 cells (Livezey et al., 2002). CLCs could inform the search for marker sequences for cell line authentication (Almeida et al., 2016).\n\nWe developed and released a subtraction database called SDB1. This sequence database consists of the human genome reference sequence, various contaminants often found in cell line sequence data, and a collection of cell line contigs called CLCs. The CLCs were created by sequencing HepG2, HuH-7, the Jurkat cells, subtracting reads that map fully and concordantly to the human genome reference, and applying de novo assembly to the remaining reads. The CLC construction process was designed to exploit the high-quality human reference genome sequence to quickly capture differences embodied in each cell line genome. Our analysis of the relative importance of the various SDB1 components indicated that the CLCs were responsible for an order of magnitude more subtraction than the suspected contaminants E. coli, Mycoplasma, and phiX.\n\nThe subtraction process reduced the computational cost that would be incurred by characterization of every read. The subtraction process removed host reads which often represented 99% of the data. The subtraction process used mapping software which imposed approximately 1% of the computational cost compared to characterization by BLAST. However, the identification of known sequences could be accomplished at lower cost using alignment-free K-mer matching software such as Kraken (Wood & Salzberg, 2014).\n\nCharacterization of the CLCs remains for future work. It is likely they capture cell-line specific chromosomal breakpoints such as those reported in HepG2 (Wong et al., 2000) and could harbor cell-line specific retrotransposed insertions. Some of the CLC sequences appear to be common to two or three of the cell lines used here.\n\n\nData Availability\n\nThe genomic and transcriptomic sequencing reads are available in NCBI; hyperlinked BioSample accessions are listed in Table S1. The subtraction database is available at GitHub (JCVenterInstitute): https://github.com/JCVenterInstitute/HumanSubtractionDB1/blob/master/SDB1.fasta.gz\n\nArchived scripts as at time of publication: http://doi.org/10.5281/zenodo.1146104 (Miller et al., 2018)\n\nLicense: GNU GPL v3.0", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJCVI staff was supported by DHS contract HSHQDC-15-C-B0059.\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\nThe authors thank Martin Hölzer for the use of data and manuscript feedback.\n\n\nSupplementary material\n\nThe following are provided as an Excel workbook (Click here to access the data):\n\nTable S1: Characteristics and accessions of human cell line sequencing data.\n\nTable S2: Human reference coverage by cell line reads.\n\nTable S3: Size statistics for Cell Line Contigs (CLCs).\n\nTable S4: Composition of the SDB.\n\nTable S5: Contribution per cell line.\n\nTable S6: Blast characterization of the largest CLCs per cell line.\n\nTable S7: Top blastn hits for JCVI nonSDB reads.\n\n\nReferences\n\nAlmeida JL, Cole KD, Plant AL: Standards for Cell Line Authentication and Beyond. PLoS Biol. 2016; 14(6): e1002476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCingöz O, Paprotka T, Delviks-Frankenberry KA, et al.: Characterization, mapping, and distribution of the two XMRV parental proviruses. J Virol. 2012; 86(1): 328–338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaly GM, Leggett RM, Rowe W, et al.: Host Subtraction, Filtering and Assembly Validations for Novel Viral Discovery Using Next Generation Sequencing Data. PLoS One. 2015; 10(6): e0129059. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDilley KA, Voorhies AA, Luthra P, et al.: The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing. PLoS One. 2017; 12(6): e0178717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaísca P, Desmecht D: Sendai virus, the mouse parainfluenza type 1: a longstanding pathogen that remains up-to-date. Res Vet Sci. 2007; 82(1): 115–125. PubMed Abstract \| Publisher Full Text\n\nHölzer M, Krähling V, Amman F, et al.: Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells. Sci Rep. 2016; 6: 34589. 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): 357–359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin YL, Liu CC, Lei HY, et al.: Infection of five human liver cell lines by dengue-2 virus. J Med Virol. 2000; 60(4): 425–431. PubMed Abstract \| Publisher Full Text\n\nLindenbach BD, Evans MJ, Syder AJ, et al.: Complete replication of hepatitis C virus in cell culture. Science. 2005; 309(5734): 623–626. PubMed Abstract \| Publisher Full Text\n\nLivezey KW, Negorev D, Simon D: Increased chromosomal alterations and micronuclei formation in human hepatoma HepG2 cells transfected with the hepatitis B virus HBX gene. Mutat Res. 2002; 505(1–2): 63–74. PubMed Abstract \| Publisher Full Text\n\nMarozin S, Altomonte J, Stadler F, et al.: Inhibition of the IFN-β Response in Hepatocellular Carcinoma by Alternative Spliced Isoform of IFN Regulatory Factor-3. Mol Ther. 2008; 16(11): 1789–1797. PubMed Abstract \| Publisher Full Text\n\nMiller JR, Harkins DM, LaPointe M: JCVenterInstitute/HumanSubtractionDB1: SendaiExperiment (Version v1.0). Zenodo. 2018. Data Source\n\nOllier L, Caramella A, Giordanengo V, et al.: High permissivity of human HepG2 hepatoma cells for influenza viruses. J Clin Microbiol. 2004; 42(12): 5861–5865. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSainz B Jr, Barretto N, Uprichard SL: Hepatitis C virus infection in phenotypically distinct Huh7 cell lines. PLoS One. 2009; 4(8): e6561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWong N, Lai P, Pang E, et al.: A comprehensive karyotypic study on human hepatocellular carcinoma by spectral karyotyping. Hepatology. 2000; 32(5): 1060–1068. PubMed Abstract \| Publisher Full Text\n\nWood DE, Salzberg SL: Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014; 15(3): R46. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30576", "date": "19 Mar 2018", "name": "Caroline C. Friedel", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors present a host subtraction database for human cell line sequencing. The purpose of this approach is subtract non-viral reads vom RNA-seq samples of virus-infected cells to improve efficiency of subsequent analyses. Their substraction database is targeted specifically towards three commonly used human cell lines: HepG2, HuH-7 and Jurkat.\nIn brief, their approach was as follows: For creation of the host subtraction database (SDB): - Perform DNA sequencing for the cell lines considered - Align reads to the human reference genome - Assemble unaligned reads into contigs The SBD was then created as a concatenation of FASTA sequences of the assembled contigs, the human reference genome, the UniVec Database (nucleic acid sequences which may be of vector origin), the PhiX genome (used as control in Illumina sequencing) and a collection of Mycoplasma genomes\nThe subtraction process was then performed by aligning reads with bowtie2 against the SDB and then removing all read pairs if at least one read in the pair could be mapped with a local alignment to the SDB.\nEvaluation of their approach was performed using RNA-seq data of the three cell lines either infected or not infected with Sendai Virus (SeV) as well as Huh-7 cells infected with Ebola or Marburg virus or not infected. They show that after host subtraction, virus reads make up the largest portion of the remaining reads. They also show that the combined runtime of first running bowtie2 and then BLAST on the remaining reads, e.g. to identify the virus, is only around 20% of the time that would be required if BLAST were run on all reads.\nWhile the article is well written and the method is well described, there are a number of issues that need to be addressed:\n1) The advantage of adding contigs assembled from DNA sequencing to the SDB is relatively small. Only between 0.19% and 1.34% of reads were subtracted in the SeV test case. However, the effort for obtaining these contigs is substantial as it requires DNA sequencing and contig assembly. Thus, the SDB cannot be easily extended to other cell lines without additional experiments. To show that this additional effort is warranted, the authors should at least show the following:\nThat removing reads mapping to the contigs before post SDB mapping has a substantial influence on the results by either resulting in reduced numbers of false positive mappings to the Sendai genome or substantially reducing runtime of the total pipeline (including SDB mapping and potentially BLAST analysis). That a similar effect cannot be obtained by augmenting the human genome by publicly available human sequence data not part of the reference genome, e.g. transcript sequences from Ensembl or RefSeq or alternatively publicly available RNA-seq data for the considered cell lines. In particular, the latter might even outperform DNA sequencing for the cell lines as assembly of reads not mapping to the human genome would result in novel transcript sequences to which RNA-seq reads map better with the (unspliced) read aligner used in this approach. Furthermore, it would allow easily extending the SDB approach to other cell lines for which RNA-seq data already is/becomes available.\n\n2) There should be some justification for the use of an unspliced read aligner, i.e. bowtie2, for mapping RNA-seq reads against the SDB, rather than a dedicated RNA-seq mapping program that also allows identifying spliced read alignments, such as STAR or HISAT2. In particular, the latter is both fast and requires relatively little memory. While removing reads for which bowtie2 identifies a local alignment likely identifies all sequences originating from the SDB sequences, there is a potential that too many reads may be removed, e.g. in case of local similarities between SDB sequences and the virus in consideration. A safer approach would be to only exclude reads that can be aligned completely or almost completely (including gaps due to splicing). Regarding this point, the authors should show that:\nthere is a substantial speedup of using local alignment with bowtie2 compared to fast RNA-seq alignment with HISAT2 or STAR that results do not differ much between the two approaches, i.e. the bowtie2 approach does neither miss many mappings a dedicated RNA-seq mapper would find nor reports many mappings not found using an RNA-seq mapper. If the latter is the case, they should show that these additional mappings missed by an RNA-seq mapper are valid mappings to the human cell lines and influence subsequent results.\n\nFinally, DNA and RNA sequencing data for the considered cell lines should be submitted to a public database to ensure full reproducibility of their approach.\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? No", "responses": [ { "c_id": "3794", "date": "12 Jul 2018", "name": "Jason Miller", "role": "Author Response", "response": "Reviewer Comment #1. This comment is focused on the cell line contigs (CLCs) that were constructed and added to the subtraction database (SDB). It notes that the CLC construction investment yielded relatively little benefit. It says the manuscript should contain at least two follow-up experiments to measure the effectiveness of SDBs constructed with and without CLCs. The additional experiments might inform those constructing SDBs on other cell lines. We agree that the manuscript should clearly convey the finding that the CLCs offered little advantage on these cells and these viruses. The manuscript already noted that the CLCs subtracted only 0.19% to 1.34% of RNA reads, providing “a small amount of mapping sensitivity.” However, the extensive treatment of CLCs in the Discussion seemed to emphasize their role. We deleted almost three paragraphs about CLCs from the Discussion. We inserted what is now the third paragraph of Discussion which includes the statement, “It thus appears that, for the cells and viruses tested, the cost of CLC construction outweighed the benefit.” With this change incorporated, it is important to note the Huh7, HepG2, and Jurkat cell lines are commonly used for viral propagation. Cell lines often have genomic abnormalities not previously reported in public databases. From this standpoint, generation of sequence data and the corresponding CLCs provides a valuable reference dataset to the community, if only to show consistency with public domain data. The first sentence of the revised Discussion now says host sequence databases provide “a critical control to ensure experimental results are attributed to the specific virus being tested.” One can question whether an essentially equivalent resource could be reproduced at lower cost. Performing that experiment would go beyond the scope of this F1000Resarch paper, which announces an existing resource that readers may download without charge. The question is certainly relevant for construction of future subtraction databases. Therefore, we modified the manuscript to raise the issue. The new third paragraph of Discussion includes the statement, “Investigators constructing subtraction databases for other human cell lines should evaluate the utility of existing public sequences prior to cell line sequencing.” It also lists types of cell lines for which sequencing could reasonably provide more benefit than it did for us. Reviewer Comment #2. This comment is focused on the mapping of RNA reads to genomic sequences. It says the process may remove too many reads because the Bowtie2 mapper is not splice-aware. It says the manuscript should demonstrate that Bowtie2 performs better than alternatives such as HiSat2. Our process employs high sensitivity by design. It uses Bowtie2 parameterized for sensitive alignments and it subtracts both RNA reads of any pair that shows any detectable alignment, including pairs with only one partial alignment to one read. We considered HiSat2 during the design phase, but we reasoned that a HiSat2-based process would be less sensitive if it only subtracted reads with partial alignments explained by splicing. One could probably design and build several alternate processes, test them on our data, and characterize all the reads that subtract differentially. Such testing might be informative, but it would go beyond the scope of this F1000Research manuscript, which describes one process that is already functional, characterized, and available. Since many readers may wonder about our choice of mapper, we modified the manuscript to point out that one could implement a splice-aware variant of our process. The new text, added to the second paragraph in the revised Discussion, says, “Our subtraction process relied on the Bowtie2 mapping software. The process could be modified to use instead a splice-aware aligner such as HiSat2. Whereas our highly sensitive process subtracts all read pairs with even one partial alignment, a splice-aware process might achieve higher specificity by subtracting only those RNA pairs with full-length alignments including spliced alignments. Being a faster mapper, HiSat2 might also reduce the computational cost of subtraction.” Reviewer Comment #3. This comment is focused on data availability of sequence reads. All of the DNA and RNA reads were previously deposited at NCBI but the fact was easy to miss with accessions being given in the supplement only. The revised text says, “The sequencing reads are available at NCBI with these SRA accessions: HepG2 DNA (SRR5296488, SRR5296494, SRR5296491) and RNA (SRR5296490, SRR5296492, SRR5296493, SRR5296495), HuH-7 DNA (SRR5297887, SRR5297975, SRR5297924) and RNA (SRR5297992, SRR5297976, SRR5297993, SRR5297994), and Jurkat DNA (SRR5294049, SRR5293982, SRR5293981) and RNA (SRR5293979, SRR5293983, SRR5295385, SRR5293984).”" } ] } ]	1	https://f1000research.com/articles/7-98
https://f1000research.com/articles/8-702/v1	21 May 19	{ "type": "Method Article", "title": "FaceSync: Open source framework for recording facial expressions with head-mounted cameras", "authors": [ "Jin Hyun Cheong", "Sawyer Brooks", "Luke J. Chang", "Sawyer Brooks", "Luke J. Chang" ], "abstract": "Advances in computer vision and machine learning algorithms have enabled researchers to extract facial expression data from face video recordings with greater ease and speed than standard manual coding methods, which has led to a dramatic increase in the pace of facial expression research. However, there are many limitations in recording facial expressions in laboratory settings. Conventional video recording setups using webcams, tripod-mounted cameras, or pan-tilt-zoom cameras require making compromises between cost, reliability, and flexibility. As an alternative, we propose the use of a mobile head-mounted camera that can be easily constructed from our open-source instructions and blueprints at a fraction of the cost of conventional setups. The head-mounted camera framework is supported by the open source Python toolbox FaceSync, which provides an automated method for synchronizing videos. We provide four proof-of-concept studies demonstrating the benefits of this recording system in reliably measuring and analyzing facial expressions in diverse experimental setups, including group interaction experiments.", "keywords": [ "facial expressions", "affective computing", "Python toolbox", "head-mounted camera", "synchronization" ], "content": "Introduction\n\nFacial expressions provide rich information about how a person is feeling, what they are thinking, and how they might act (Russell & Fernández-Dols, 1997). Facial expressions captured the interest of early theorists (Darwin, 1872; James, 1884) and remain a popular method for noninvasively studying behavioral displays of emotions. Pioneering work by Paul Ekman established the Facial Action Coding System (FACS; Ekman & Oster, 1979), which provided a reliable coding system of different facial muscles referred to as action units (AUs) and allowed facial expressions to be compared across people and cultures (Matsumoto & Ekman, 1989; Matsumoto et al., 2009).\n\nExtracting facial expression information through FACS coding, however, can be a labor intensive and time-consuming process. Becoming a certified FACS coder not only requires 100 hours of training (“Paul Ekman Group,” 2017) but even a well-trained coder may need over an hour to code a single minute of video (Cohn et al., 2007). In addition, manual coding inevitably exposes the data to human errors in coding or biases, therefore requiring researchers to collect ratings from more than one coder, further complicating the process.\n\nAs an alternative to manual FACS coding, automated measurements of facial expressions can substantially reduce the amount of time required to extract facial expression information. One technique, known as facial electromyography (fEMG), measures the electrical impulses associated with facial muscle movements. With fEMG, researchers can continuously measure the activity from muscle groups associated with facial AUs, such as the zygomaticus major and corrugator supercilii muscles, at a high sampling rate. However, fEMG requires a separate electrode for each facial muscle group, which means that only one or two muscle groups are recorded simultaneously in practice (Fridlund & Cacioppo, 1986; Wolf, 2015). Even so, the recordings may include signals from not only the target muscle but also overlapping or nearby muscles making it difficult to distinguish the precise activity of the target muscles (Cohn & Ekman, 2005). Moreover, this technique does not scale well to recording multiple participants interacting in a social experiment as recordings can be sensitive to movement artifacts and having wires attached to one’s face can be unnatural and obtrusive.\n\nAutomated extraction of facial expression information from face video recordings have emerged as a promising alternative that offers quick, continuous, and simultaneous measurement of multiple facial muscle movements without manual coding. Advances in computer vision and machine-learning techniques (e.g., kernel methods, deep learning) and large-scale data collection have facilitated the development of models that learn to transform pixels from videos into predictions of facial AUs and emotional facial expressions (Amos et al., 2016; Littlewort et al., 2011; Michel & Kaliouby, 2003; Susskind et al., 2007). Consequently, this has facilitated an explosion in scientific articles related to facial expression analysis with a sixfold increase over the past decade.1\n\nThis automated approach has offered much insight into human behavior. Automated extraction of facial expressions has been used to predict a wide range of behaviors including task engagement (Whitehill et al., 2014), automobile accidents (Ahn et al., 2010), effectiveness of advertisements (McDuff et al., 2015), and online purchase behaviors (Ahn et al., 2008). Cultural differences in facial behavior have also been examined at a larger scale spanning more than 31 countries (McDuff et al., 2017) as well as sex differences in smiling (McDuff et al., 2017). Facial expressions have also shown promise in clinical settings to quantify symptom severity in neuropsychiatric disorders such as Schizophrenia and Parkinson’s disease (Bandini et al., 2017; Hamm et al., 2011) and depression (Girard et al., 2015), and also for detecting evidence of malingering pain symptoms (Bartlett et al., 2014).\n\nThe acquisition of high temporal and spatial resolution of facial expressions in laboratory environments, however, has remained challenging. Popular solutions such as webcams, tripod-mounted cameras, and pan-tilt-zoom (PTZ) cameras (Figure 1A–C) require compromising between cost, flexibility, and reliability. In this article, we demonstrate the feasibility of head-mounted video cameras as an alternative to standard recording setups. We provide step-by-step instructions on how to build affordable head mounts using readily available materials and minimal technical expertise. We demonstrate how the head-mounted camera can provide reliable recordings that is invariant to head-rotation and can be flexibly used in a variety of experimental settings such as stimulus based tasks, natural viewing of videos, and social interactions. We also introduce the FaceSync toolbox which can be used in conjunction with head-mounted cameras to automatically synchronize videos in time based on audio. Overall, we provide a unique solution for recording facial expressions that is affordable, adaptable to different experimental setups, and reliable in recording an unobstructed view of the face.\n\n(A). Webcam setup with a camera positioned above the monitor. (B). Tripod-mounted camera setup placed facing the subject. (C). PTZ camera setup with the camera installed above the TV screen in a dedicated room. (D). Head-mounted camera built according to build instructions in the Underlying data. (E). Use of head-mounted camera in social interaction experiment.\n\nWhen choosing a framework for recording videos of facial behavior in lab settings, researchers must consider a variety of factors including affordability, adaptability to different experimental settings, integration with other devices, and recording reliability. In this section we survey and summarize the strengths and shortcomings of popular setups including webcams, tripod-mounted cameras, and PTZ cameras (Table 1).\n\nThe most readily available and easy-to-implement option is to record from computer webcams. External webcams with good image resolution can cost about $100 but most modern laptops and computers come with pre-installed webcams integrated on top of the screen providing a low profile setting less likely to capture the attention of participants. Webcams are effective for event based experiments in which facial expressions can be recorded using the same computer hosting the task. Webcams can be triggered and controlled via programming languages providing a scalable solution for recording social interactions through video call setups. Such interactions, however, may not provide the same experience as live face-to-face interactions (Sherman et al., 2013; Shin et al., 2017). Moreover, integrated webcams can be limited in temporal resolution as they rely on shared computing resources and use variable frame rates between 24 to 30 frames per second (fps) to optimize the use of computer resources. In addition, the fixed position of webcams and the distance between the camera and the face allow bodily movements touching the face, head rotations, or out-of-plane head motions to cause difficulties in capturing and extracting facial expressions from the videos. Therefore, it is difficult to consider webcams as a robust and reliable solution to recording facial expressions despite being easy to use and cost-effective.\n\nTripod-mounted cameras cost around $1,000 for production-quality camcorders and can provide high-resolution recordings at faster frame rates. Tripod-mounted cameras can be manually moved or adjusted by the experimenter to account for subject movements if the experimenter can be present during the experiment at the cost of increased conspicuousness. Tripod-mounted cameras can be easily installed and removed to accommodate different experimental settings allowing for flexibility in experimental setups. They can moved to different experimental environments and adjusted to different heights and angles to best capture facial behaviors. Scalability, however, is limited as adding additional cameras remains expensive and synchronizing across multiple cameras can be challenging as time-code or TTL (transistor-transistor-logic) pulse triggering capable camcorders are often more expensive.\n\nThe PTZ camera setup provides researchers centralized control over cameras that can be rotated or zoomed to account for subject movement. PTZ camera setups require a dedicated experiment room with cameras installed and an adjacent console room where experimenters can monitor incoming video feeds and control the camera. Central management of cameras can facilitate integration with other softwares or triggering the cameras to record simultaneously. The installation of cameras to corners of ceilings distant from the participant allows cameras to be less conspicuous. However, this forces participants to stand or sit in particular locations in the room and renders the setup particularly susceptible to head rotations or occlusions of the face from body gestures unless multiple cameras are installed. It is the least flexible option because changing camera locations or installing additional cameras would require additional construction. The PTZ camera setup can therefore be the best option to minimize participants’ attention to the camera but at the cost of increased possibility of artifacts and reduced adaptability to other experimental setups.\n\nOverall, webcams, tripod-mounted cameras, and PTZ cameras do not provide an optimal solution for recording facial expressions. They commonly suffer data loss due to out-of-plane head motions and head rotations (Cohn & Sayette, 2010; Lucey et al., 2011; Werner et al., 2013), although developing algorithms robust to partial face occlusions is an active area of research (Zhang et al., 2018). Another common challenge pertains to the temporal precision in the alignment of simultaneous recordings between cameras and to the stimuli. In the next section, we propose head-mounted cameras as an affordable, scalable, and flexible solution that can provide reliable recordings and can be easily synchronized with experimental stimuli and across recordings from multiple cameras.\n\n\nHead-mounted cameras\n\nA head-mounted camera recording system (Figure 1D) provides a unique solution to the limitations of the surveyed methods. It is a highly adaptive system that can be used for different experimental setups ranging from computer-based tasks to multi-person social interaction experiments (Figure 1E). The head-mounted camera consists of a single camera attached to the head of the participant using lightweight head-gear. This setup removes the impact of head rotation or body movements obstructing the view of the face leading to face detection failure and increases reliability. However, as a result, it cannot detect bodily movements and gestures, unless additional cameras are installed, or head orientation information, unless additional gyro sensors are attached. It is minimally cumbersome other than the weight of the gear and protrusion from the head-gear, and it can be positioned below the line of sight of subjects, allowing the wearer to view a monitor in computer based tasks or make eye contact and track others’ facial expressions in social interaction tasks.\n\nCommercial head-mounted cameras are often used in motion capture studios and remain expensive with costs ranging from $2,500 - $20,000 for a complete camera and mount setup2. However, assembling a head-mounted camera setup in the lab can be an affordable alternative option that requires minimal engineering expertise or expensive equipment. Action cameras, such as the GoPro are well-suited for this purpose as they are inexpensive ($150 - $400), small in size, and lightweight. We provide step-by-step assembly instructions for building a head-mount for GoPro cameras in the Supplementary Information (see Underlying data) (Cheong et al., 2019) along with a parts list and blueprint files to 3D print other parts. This allows researchers to easily construct their own head-mounted camera setup for less than $700 (including the camera)3.\n\nAll video recording devices require a method to temporally align the video recordings to the experimental task. As mentioned earlier, some devices such as webcams or PTZ cameras can be controlled or triggered from the experiment computer to start and stop recording during the paradigm. Camera setups that are not directly connected to the experiment computer, including head-mounted devices, require an alternative method that is accurate and efficient for aligning the videos to experimental events.\n\nThe traditional ‘clap’ method used in the film industry uses a sharp, loud sound at the beginning of the clip that allows multiple videos to be aligned to the resulting spike in the audio waveform. This audio-based synchronization method usually requires opening each video for manual inspection of the sound waveform and incrementally shifting the audio until the echo, which indicates phase misalignment, is eliminated. Humans are highly accurate in detecting and distinguishing audio offsets down to several milliseconds (ms), but manually synchronizing each video is labor intensive and can introduce unsystematic noise in the alignment (Litovsky et al., 1999; Shrstha et al., 2007).\n\nTo facilitate the synchronization of videos, we developed FaceSync, an open-source Python toolbox, to automatically synchronize video recordings based on audio. In stimulus-based experiments, it requires a short audio segment to be played at the beginning of the experiment, which is recorded by the camera. Based on this shared audio, the toolbox can align the video to the beginning of the experiment finding the optimal alignment with the original audio segment. In unstructured social interaction experiments, multiple videos can also be aligned to a target video. The toolbox offers a sliding window search to find the offset that maximizes the correlation between two audio signals (Figure 2) and a fourier transform based cross-correlation method. The FaceSync toolbox supports both Python versions 2 and 3 can be downloaded from our github repository (https://github.com/cosanlab/facesync).\n\n(A, B) Portion of audio is selected from target audio (red waveform top panel) which is compared to a portion of the sample audio selected in a sliding temporal window (blue waveform bottom panel). Correlation similarity is calculated at each window and the offset is determined to be the temporal window that maximizes similarity between the two audios. (C). Graphical interface in FaceSync toolbox to manually align two audios. One audio file can be shifted in time using the sliders until the two waveforms are aligned. Alignment can be inspected visually by examining the waveform plots and by listening to the combined audio.\n\n\nProof of concept validation of head-mounted cameras\n\nIn the following sections, we demonstrate that the head-mounted camera recording system provides a robust way to record facial behavior in laboratory experiments. In Study 1, we show that the head-mounted cameras can reliably record facial behaviors invariant to head rotation. In Study 2–4, we demonstrate the flexibility of using head-mounted cameras in multiple experimental setups including an event-based paradigm (Study 2), naturalistic video watching paradigm (Study 3), and a social interaction paradigm (Study 4). In these three proof of concept experiments, we also compare the performance of the FaceSync software in synchronizing recordings in comparison to the manual alignment method.\n\nStudy 1: Face detection with head rotation.\n\nMethods. To examine the impact of head rotation on face registration, we recorded the face of one male participant (author J.H.C.) using a webcam and our head-mounted camera. In each recording session, the participant rotated the head 90 degrees left, returned to center, 90 degrees to the right, then returned to center. The head-mounted camera used a GoPro Hero4 camera to record 1280 x 720 resolution videos at 30 frames per second (fps). The webcam recording used the integrated camera on a Macbook Pro Retina laptop at 1080 x 720 resolution at approximately 30fps. Facial expressions were extracted using the iMotions Emotient FACET engine (iMotions Biometric Research Platform 6.0, 2016)4, which provides face registration success, landmark positions, AU predictions, and emotion predictions.\n\nResults. Face recording using the head-mounted camera retained a continuous view of the entire face without face detection failure regardless of face rotation (Figure 3A top row). In contrast, the webcam face recording resulted in face detection failure when the head was rotated (Figure 3B right panel) which subsequently resulted in failure to extract facial expression predictions.\n\n(A, B) Top row images show the face recordings when facing forward and the right panel image shows the face when facing left. Graphs in the middle row show face detection success at each frame. Graphs in the bottom row shows neutral face expression predictions. In (A), facial expressions are predicted in all frames in contrast to (B) where face detection and facial expressions predictions fail when the head is rotated away from the camera.\n\nFace detection success was 100% of the video duration in the head-mounted camera recording compared to 75% in the webcam recording due to face detection failure when the face was turned (Figure 3A, B middle row). Facial expression predictions (e.g., neutral face, Figure 3A, B bottom row) in the webcam recording also failed for 25% of the video during the head rotation while prediction from the head-mounted camera recording was unaffected.\n\nDiscussion. Comparing face recordings from a webcam and a head-mounted camera, we demonstrate that the head-mounted camera provides a more reliable and continuous recording of the face invariant to head rotation. This is important as face expression software is unable to make predictions about facial expressions when it is unable to register a face. Although the head-mounted camera is invariant to head rotations, the head position is currently not tracked. Future work might add additional sensors to monitor head position dynamics. Overall, we demonstrate that the head-mounted camera can prevent data loss due to body and head rotations that can readily occur in most experimental settings without strict restriction of participants’ natural movements.\n\nStudy 2: Recording facial expressions to event-based stimuli\n\nThis experiment demonstrates the use of head mounted cameras in recording facial expressions to time-locked stimulus presentations. Performance of automatic video alignment using FaceSync in comparison to manual adjustment is also provided.\n\nMethods. One male participant (author J.H.C.) viewed 10 positive images (e.g., kittens, puppies) and 10 negative images (e.g., injured bodies and faces) presented in MATLAB using Psychtoolbox version 3.0.12 (Brainard, 1997; Pelli, 1997) and made deliberate facial expressions concordant with the valence of the image. Each image was presented for two seconds with jittered inter-trial intervals (ITI) of 4, 6, and 8 seconds (mean ITI = 5.4 seconds). The 20 images were selected from the IAPS picture database (Lang et al., 2008). Facial behavior was recorded using a head-mounted GoPro Hero4 camera in 1,280 × 720 resolution at 30fps.\n\nAudio offset was determined both by manually synchronizing the recording using the FaceSync AudioAlign graphical interface and automatically using the FaceSync alignment function. The audio sample used for synchronization (synctune.wav) was a two-second harmonic tune constructed with sine waves at different frequencies. Four independent raters (including author J.H.C.) incrementally shifted the extracted audio in 1ms precision to the target audio using AudioAlign while listening to the shifted sound to minimize echo artifacts as well as visually checking the two waveforms for misalignment. For automatic alignment, we used the FaceSync sliding window correlation function (find_offset_corr) to detect the offset that maximizes the correlation similarity between the two audios. Alignment results from additional test videos that are not included in the face expression analysis were also tested for alignment and are reported as supplementary tests in Table 2. The video was trimmed according to the calculated offsets and facial expressions were extracted using the iMotions Emotient FACET engine (iMotions Biometric Research Platform 6.0, 2016).\n\nAudio synchronization performance comparison between manual alignment and automatic alignment with FaceSync algorithm. Offsets indicate the duration in seconds from the original file that needs to be trimmed to be aligned to the target audio. One standard deviation shown in parentheses. Supplementary tests include audios from additional videos recorded for testing alignment, but are not part of Studies 2 to 4.\n\nResults. An independent-samples t-test was conducted to compare positive facial expressions while viewing positive and negative images. Evidence of positive facial expressions while viewing positive images (M = 8.55, SD = .52) was significantly greater than the evidence while viewing negative images (M = 0.59, SD =1.53; t(18) = 15.58, p < 0.001; Figure 4A). Evidence for disgust facial expression while viewing negative images (M = 3.02, SD = 1.41) was significantly greater than the evidence while viewing positive images (M = .25, SD = .77, t(18) = 5.45, p < 0.001, Figure 4B).\n\n(A) Evidence of smiling to positive (blue) and negative (red) images. (B) Evidence of disgust facial expression to positive (blue) and negative (red) images. Shaded error bars indicate standard error of the mean.\n\nAudio alignment results are reported in Table 2. The average difference between manual audio alignment by four different raters and the FaceSync automatic algorithmic alignment was -.002 seconds (SD = .004).\n\nDiscussion. In Study 2, we demonstrate the feasibility of using head-mounted cameras to record facial expressions in response to time-locked stimuli. The participant displayed positive facial expressions (i.e. smiling) to positive images and disgust facial expressions to negative images which was accurately retrieved from the analysis. Facial expressions were successfully linked to the stimuli that elicited the response by accurate alignment of the face recording to stimulus timing. Only a small difference was observed between the offsets determined automatically and manually. This study demonstrates the feasibility of using a head-mounted camera setup for standard computer-based experiments and that facial expressions can be linked with the stimuli that elicited the response with high temporal precision.\n\nStudy 3 and 4: Watching and discussing naturalistic stimuli together\n\nTo demonstrate the feasibility of using the head-mounted camera setup to simultaneously record facial expressions from several individuals, we recorded individual facial expressions of a group while they watched a video together in Study 3. Subsequently in Study 4, we recorded facial expressions of the group members while they discussed the contents of the video. We compare the facial expression behavior of each participant to one another with the expectation that participants would show synchronization of facial expressions in both conditions but to a greater extent in the movie watching experiment. Audio alignment offsets determined by manual and automatic alignment are compared.\n\nMethod. In Study 3, we measured the facial expressions of a group (N=5, 20% Female) watching a video, Big Buck Bunny (“Big Buck Bunny,” 2008). In Study 4, the group freely discussed the content of the video. Each person’s facial behavior was recorded by their head-mounted GoPro Hero4 camera at 120fps and at 1,920 x 1,080 resolution.\n\nIn the Study 3, each face recording was aligned to the audio of the movie. In Study 4, each face recording was aligned to the audio of a single participant whose recording began the latest. In both studies, all cameras recorded the audio in the environment simultaneously, which allowed them to be aligned based on the shared audio. Each recording was aligned manually by four independent raters (including author J.H.C.) using the FaceSync AudioAlign graphical interface and automatically using the sliding window correlation alignment function (find_offset_corr). Differences in offset measured by the two methods were submitted to a one sample t-test to assess whether there was any difference between the two methods. After alignment, videos were trimmed using the FaceSync trim function.\n\nFacial expressions, including AU activations and emotion predictions, were extracted using the iMotions Emotient FACET engine (iMotions Biometric Research Platform 6.0, 2016) and subsequently downsampled to 1hz. To assess the similarity of the affective experience, we calculated intersubject synchrony (ISC). This technique has been used in fMRI analysis to identify signals that are common across participants when watching naturalistic stimuli such as movies and listening to stories (Hasson et al., 2004; Honey et al., 2012; Nummenmaa et al., 2012; Stephens et al., 2010). For each experiment, ISC for joy facial expressions was calculated using pairwise correlation similarity of participants’ predicted joy time-series. To determine whether the group was synchronizing in their smiling greater than chance, we calculated a one-sample t-test over all pairwise correlations to test whether the synchronization was significantly different from zero. In addition, we used a paired-sample t-test to assess whether the ISC was significantly different between the viewing (Study 3) and the discussion (Study 4). All t-tests were conducted on Fisher r-to-z transformed correlations.\n\nResults. Overall, we found evidence that participants were having a similar affective experience while watching the video (Figure 5). Average ISC was r = .40 (SD=0.08), t(9) = 13.89, p < 0.001. Participants also displayed synchronized facial expressions while discussing the video, r = .20, (SD=.16), t(9) = 3.75, p = 0.004. However, there appeared to be greater ISC of the joy facial expression viewing the show compared to discussing it afterwards, t(18) = 3.53, p = 0.002. This is likely because the joy facial expression was noisier while participants were talking and participants were not always in agreement with each other.\n\n(A) Pairwise intersubject similarity matrix for joy while watching the video is shown on the left panel. Three subject videos are shown on the right with corresponding joy evidence predictions. (B) Pairwise intersubject similarity matrix for joy while discussing the video is shown on the left panel.\n\nThe average difference between the automated offset detection and the manual offset search was -.005 seconds (SD = .027; Table 2) for the movie watching videos and .011 seconds (SD = .024; Table 2) for the movie discussion session. Across all three studies and including additional supplementary videos that were similarly evaluated (see Table 2 footnote), overall differences in offsets calculated manually and automatically was .001 seconds (SD = .022) and was not significantly different (t(50) = .46, p = .65).\n\nDiscussion. In these two studies, we observed a relatively high level of synchronization in affective experiences across participants while viewing and subsequently discussing a video. Speaking appears to decrease the sensitivity of ISC, likely as a result of the added noise from the mouth movements while speaking. The FaceSync toolbox can accurately align videos together even when the audio recordings are non-uniform due to location of the camera position and multiple people talking at the same time. Overall, these studies demonstrate the flexibility of using head-mounted cameras to record facial behavior in naturalistic experimental paradigms such as watching a movie and also in social experiments, in which participants interact with each other.\n\n\nDiscussion\n\nIn this paper, we provide evidence that head-mounted cameras offer a robust, flexible, and affordable solution to recording facial expressions in the laboratory. In four proof of concept studies, we first demonstrate that the head-mounted camera yields reliable face recordings that allow facial expression analysis irrespective of head motion. Second, we demonstrate the flexibility of using head-mounted cameras across different experimental settings from traditional stimulus based experiments to group social interaction experiments. By using the FaceSync toolbox to align recordings to stimulus onsets, facial expressions were successfully linked to the events that triggered facial behavior, such as increased positive facial expressions in response to viewing positive images, and increased disgust facial expressions in response to viewing negative images. In addition, facial expressions were more synchronized when watching a video compared to when discussing the video. Most importantly, we demonstrate that the FaceSync toolbox can accurately and automatically align video recordings with comparable accuracy with manual alignment. Together, these results demonstrate that the head-mounted camera setup offers a reliable and robust method for recording facial expressions in a variety of experimental settings and can scale to n-person social experiments.\n\nThe head-mounted camera setup can still be improved in several ways. For example, lighting conditions are important in face detection such that poor luminance of the face or extreme backlights can lead to face detection failures. Researchers should be aware of this issue and should avoid situations where ceiling lights or window sunlight in the background decrease face luminance. LED lights can be attached to the head mount to control for these issues by providing equal luminance of the face.\n\nAnother potential improvement is the weight and size of the camera. The weight of the camera that pulls the headgear downward can be a source of discomfort if worn for extended periods of time. At the time of construction, the camera (i.e., GoPro Hero4 Black with camera, memory card, and lens cover) weighed 92 grams but now more recent models (i.e., GoPro Hero5 Session) weigh only 74 grams. Small reductions in weight can lead to increased comfort as the tugging force is significantly reduced based on the length of the headset. Researchers can take advantage of newer and lighter cameras as they become available as the 3D printable camera mount provided with the blueprints is compatible with other cameras.\n\nIn summary, we hope that these tools can benefit other researchers and further accelerate facial expression research. We anticipate that this framework will aid in improving our understanding of how emotions are experienced in naturalistic settings and how emotions surface and influence social interactions. In addition, we hope that these tools can aid researchers in developing new models of complex emotions such as regret, guilt, and gratitude. We look forward to a new era of facial expression research that examines facial behavior in both controlled and naturalistic settings to yield a robust and holistic understanding of how facial expressions reflect our thoughts, emotions, and future behavior.\n\n\nEthical statement\n\nThis study was approved by the Institutional Review Board at Dartmouth College and written consent from all participants was obtained. Informed written consent was obtained for all individuals whose full faces are shown in the figures or in the videos in data repository.\n\n\nSoftware availability\n\nSource code for FaceSync toolbox available from: https://github.com/cosanlab/facesync\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.2638335 (Cheong et al., 2019)\n\nLicense: MIT\n\n\nData availability\n\nOpen Science Framework: FaceSync: Open source framework for recording facial expressions with head-mounted cameras. https://doi.org/10.17605/OSF.IO/B2NUA (Cheong et al., 2019)\n\nThis project contains the following underlying data:\n\n\n\n- Analyses folder containing: FiguresForFaceSync_OSF.ipynb (Jupyter Notebook with scripts to completely recreate the analyses and figures)\n\n- Data folder containing: Raw data necessary to recreate the analyses, such as videos, extracted facial expression information, and sounds used for synchronizing the videos (in .txt, .csv, .wav and .MP4 formats)\n\n- Supplementary Information folder containing: FaceSync_build_information.pdf (detailed instructions on how to build the FaceSync head-mounted recording system, including parts List and build instructions, and Supplementary Tables 1 and 2)\n\n- Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nAuthors thank Seth Frey, Aimee Shu-Chen Sung for participating in the demonstrations and Eshin Jolly for feedback on this manuscript. 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Publisher Full Text" }	[ { "id": "52689", "date": "10 Sep 2019", "name": "Elliott Ross", "expertise": [ "Reviewer Expertise Behavioral Neurology", "Affective Prosody", "Memory", "Emotions", "Facial Expressions (perception and motor physiology)" ], "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 “proof of concept” publication that introduces a novel head-mounted camera apparatus for obtaining spatially accurate videos of facial expressions that is able to compensate for unwanted head movements that often occur during research projects, especially those that involve spontaneous facial expressions. The apparatus uses a small, light-weight and relatively inexpensive Go-Pro video camera (30 fps, 1,280 x 720 pixels) that is mounted on a rectangular(light-weight frame work that in turn has a head mount that rests on top of the head and over the ears so that it does not block or interfere with the facial muscles that give rise to forehead expressions (see Figure 1, panels D and C, and Figure 3, panel A).\n\nThe authors provide directions for construction of the apparatus with blueprints and provide a means for the facial expression videos to be synchronized with audio recordings for accurate off-line analyses (FaceSync). In addition, the authors present 4 brief “proof of concept” experiments that demonstrate the utility of their apparatus and FaceSync. The most important is Study 1, in which the subject moves his head 90 degrees to the left and then 90 degrees to the right of center. The video was then analyzed off-line using the iMotion Emotient FACET engine that provides “face registration success, landmark positions, AU predictions and emotion predictions.” Face detection was 100% successful for the entire video. In comparison, using a webcam (stationary) video recording for the same sequence, only 75% of the video was successfully analyzed because the iMotion Emotient FACET engine failed to detect the face during head rotation.\nThis publication makes a major contribution to the quantitative analysis of facial expressions that use video recordings. To date, research videos of facial expressions are obtained by using a fixed camera set-up that cannot compensate for spontaneous head movements, i.e. a computer webcam, a tripod-mounted camera or a pan-tilt-zoom camera (see Figure 1, panels A, B and C). Although software exists that can partially compensate for head movement in a given plane, it is mathematically very difficult, if not impossible, to compensate for head movements that occur over multiple planes, especially rotatory head movements, when attempting to quantitatively analyze facial expressions using computer software techniques. Thus, the head-mounted camera apparatus will allow researchers to obtain better and more complete quantitative data regarding facial expressions. My only suggestion to the authors is that they might consider adding an adjustable counter weight to the back of their apparatus to make the frame more comfortable for the subject.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "60016", "date": "05 Mar 2020", "name": "Sylwia Hyniewska", "expertise": [ "Reviewer Expertise Computer science and psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes head-mounted camera systems as the most appropriate state-of-the-art recording devices for facial expression analysis. Two drawbacks of such setups are emphasised by the authors: the cost of commercialised headmounts and synchronisation of recordings with studied stimuli.\nFirst, the authors provide step-by-step assembly instructions to enable researchers to easily construct their own head-mounted camera setup as an affordable alternative option that requires minimal engineering expertise.\n\nSecond, the authors introduce FaceSync, an open-source Python toolbox that they developed to automatically synchronize video recordings based on audio cues. A short audio segment can be played at the beginning of the experiment by e.g. a stimuli presenting software and be recorded by the head mounted camera. The toolbox allows the alignment of the video to the sound produced by the experimental software. In unstructured social interaction recordings, multiple videos can be aligned to a target video. The toolbox offers a sliding window search to find the offset that maximises the correlation between two audio signals and a fourier transform based cross-correlation method. To test that the head-mounted camera recording system provides a robust way to record facial expressions, the authors ran several studies with users.\n\nNowadays, however, facial expression analysis is not limited to fixed cameras and webcams. The authors describe perfectly the added value of head mounted cameras for (automatic) facial expression analysis but do not describe the current state of the art in terms of comparable hardware and software. Given fast progress, e.g. in immersive technologies, a variety of solutions exist. The authors should consider discussing the expensive commercial options (e.g. Google glasses with Emotiv SDK and OpenCV for visual processing) and the open access options that do exist, e.g. for video alignment and facial expression recording from similar devices. If no user study can be done on similar hardware and software for comparison, please provide sufficient information in the state of the art and discussion sections.\nPlease consider emphasising the added value of the article/framework in the title and abstract.\nPlease make it clearer whether FaceSync is a synchronisation system (based on audio) as described in the manuscript or a framework for recording facial expression, as in the title.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-702
https://f1000research.com/articles/8-699/v1	21 May 19	{ "type": "Opinion Article", "title": "Gut microbiome-Mediterranean diet interactions in improving host health", "authors": [ "Ravinder Nagpal", "Carol A. Shively", "Thomas C. Register", "Suzanne Craft", "Hariom Yadav", "Ravinder Nagpal", "Carol A. Shively", "Thomas C. Register", "Suzanne Craft" ], "abstract": "The gut microbiota plays a fundamental role in host health and disease. Host diet is one of the most significant modulators of the gut microbial community and its metabolic activities. Evidence demonstrates that dietary patterns such as the ‘Western diet’ and perturbations in gut microbiome (dysbiosis) have strong associations with a wide range of human diseases, including obesity, metabolic syndrome, type-2 diabetes and cardiovascular diseases. However, consumption of Mediterranean-style diets is considered healthy and associated with the prevention of cardiovascular and metabolic diseases, colorectal cancers and many other diseases. Such beneficial effects of the Mediterranean diet might be attributed to high proportion of fibers, mono- and poly-unsaturated fatty acids, antioxidants and polyphenols. Concurrent literature has demonstrated beneficial modulation of the gut microbiome following a Mediterranean-style diet in humans as well as in experimental animal models such as rodents. We recently demonstrated similar positive changes in the gut microbiome of non-human primates consuming a Mediterranean-style diet for long term (30 months). Therefore, it is rational to speculate that this positive modulation of the gut microbiome diversity, composition and function is one of the main factors intermediating the health effects of Mediterranean diet on the host. The present perspective discusses the evidences that the Mediterranean diet induces gut microbiome modulation in rodents, non-human primates and human subjects, and discusses the potential role of gut microbiota and microbial metabolites as one of the fundamental catalysts intermediating various beneficial health effects of Mediterranean diet on the host.", "keywords": [ "fiber", "gut microbiota", "Mediterranean diet", "monkey", "non-human primate", "short-chain fatty acids", "western diet" ], "content": "Introduction\n\nDiet, gut microbiome and chronic diseases like obesity, diabetes, cancer and aging-related diseases such as Alzheimer’s disease are closely associated. Our dietary habits have a strong effect on our gut microbiome and metabolism. Unsolicited perturbations in gut microbiota diversity and composition (gut dysbiosis) are key elements underlying chronic diseases including several low-grade inflammatory disorders of human gastrointestinal tract1. Specifically, low consumption of dietary fibers is known to induce long-term changes in the gut microbiome that are associated with low production of beneficial microbial metabolites, i.e., short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate involved in the modulation of host immune and inflammatory health status2. Epidemiological investigations have reproducibly found that the ‘Western-style’ dietary habits (typically characterized by low consumption of fruits, vegetables, salads, fish and mono- and poly-unsaturated fatty acids such as fish and olive oil, and high consumption of simple sugars, saturated fat, red meat and processed foods) as one of the main perpetrators for the rising worldwide incidence of chronic diseases3. From that perspective, a dietary-pattern intervention targeting gut microbiome can provide an effective avenue for the prevention and treatment of such chronic diseases.\n\n\nMediterranean diet: the intangible cultural and nutritional dietary pattern\n\nPrompted by this evidence, a move towards a Mediterranean-style diet is exemplified as not only a prudent choice of lifestyle but also as a scientifically accepted mechanism that is able to yield the benefits for management of several human disease pathologies and an overall improvement of health and well-being. In 2013, the Mediterranean diet (hereafter, MD) was also enrolled on the “Representative List of the Intangible Cultural Heritage of Humanity” by the United Nations Educational, Scientific and Cultural Organization (UNESCO)4. In 406 B.C., Hippocrates, the father of medicine, had already stated: “Let food be your medicine and medicine be your food”. Strikingly, this 20-centuries-old statement has been clearly and consistently validated by the ever-mounting literature indicating that the dietary habits can modulate predisposition to various human gastrointestinal, metabolic, cardiovascular and systemic diseases. A Mediterranean-style diet typifies a nutritionally balanced diet, characterized by intake in high amounts and frequency of important sources of fibers (cereals, vegetables, legumes, fruits and nuts) and chemical ingredients with anti-oxidative properties (vitamins, flavonoids, phytosterols, minerals, terpenes and phenols) (Table 1)5. In addition, high proportions of oleic acid, polyphenols and unsaturated fatty acids delivers significant anti-atherogenic and anti-inflammatory properties (Table 2)6. Studies have demonstrated that switching to a Mediterranean-style diet demonstrates amelioration in serum inflammation biomarkers as well as their gene expression profile (nutrigenomics), not only in healthy subjects7. but also in patients with obesity, type-2 diabetes and Crohn's disease (Table 2)8–15. Such dietary habit changes are also accompanied by specific changes in the population level of several gut microbial groups6,16–20. Although the association of diet-microbiome interactions with the host health and disease status remains to be comprehended by means of all-inclusive and mechanistic epidemiological and omics investigations and specific disease related pathologies; however, advancements in novel hypotheses and postulations have already exceeded over and beyond merely a speculation stage. In this context, while concurring that MD represents a promising, efficacious and holistic approach to maintain/restore host heath, it is equitable to believe that (many of) these health benefits of MD are mediated via modulation of host gut microbial clades and their metabolic functions (Figure 1).\n\nReferences are shown in square brackets.\n\n\nMD, gut microbiome and host health\n\nThe human gut microbiome is comprised of tens of trillions of microbial cells. The association of gut microbiome dysbiosis with various dramatically rising human diseases such as obesity, type-2 diabetes and aging-related diseases like Alzheimer’s disease spurs urgent need for devising effective and safe treatments using gut microbiome modulators. Our dietary practices have an immense effect on many features of gut microbes. One of the primary functions of gut microbiome is to metabolize dietary ingredients in a way that the products of this biotransformation (e.g., SCFAs, vitamins, bioactive derivative compounds and modified plant flavonoids) can benefit various aspects of human health and metabolism51. Mediterranean-style diets are considered nutritionally balanced and scientifically recommended dietary pattern (Table 1)5 that are beneficial for the prevention and/or treatment of obesity, type-2 diabetes, inflammatory disorders and cardiovascular diseases (Figure 1 and Table 2)8,10,11,22,52–54. On the other hand, consumption of Western-style diets (mainly omnivore-type diets) may lead to a gut microbiome composition that is more associated with several diseases55. This increasing understanding about the importance of diet-microbiome interactions and their strong influence on human health has led to a novel and timely concept of exploring and developing ‘microbiota-directed’ foods’ that can function by modulating the functional spectrum of the gut microbial community, providing precursor substratum for microbial biotransformation to bioactive molecules beneficial for the host health, or a combination of both of these approaches56. This could provide novel and potential ways for improving host health by fostering/supporting healthy gut microbial communities, restoring the loss of microbial diversity associated with Western-style diets, and ameliorating the structural and functional dysbiosis associated with various human diseases. One example of this microbiota-directed food is ‘prebiotic’, which is defined as “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon/gut57. Indeed, a food can include one or more prebiotics (e.g., plant fibers, inulin, chicory, galacto-oligosaccharides, and others) capable of being processed by selective array of commensal/ beneficial gut microbes, leading to the production of beneficial metabolites like SCFAs. These SCFAs act as key mediators of the beneficial effects of such fibrous diets on host health51,58.\n\nGLP-1, glucagon-like peptide 1; HDL, high-density lipoproteins; IGF, insulin-like growth factor; LDL, low-density lipoproteins; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; PYY, peptide YY; SCFAs, short-chain fatty acids; VLDL, very low density lipoproteins.\n\nHigher adherence to MD has been found to be associated with reduced incidences of several chronic diseases, such as obesity, type-2 diabetes, metabolic syndrome, gastrointestinal cancer, cardiovascular diseases, fatty liver diseases, chronic kidney diseases and neurodegenerative diseases like Alzheimer’s disease (Table 2)59–63. Based on different studies, the lowering effects of MD against the incidence of these disorders have been attributed to different MD dietary constituents derived from fruits, vegetables, lean meat, nuts and grains, fibers, mono- and poly-unsaturated fatty acids, vitamins, polyphenols, and antioxidant (Table 2)64–66. Considering that the pathology of the above-mentioned diseases involves a dysbiotic gut microbiome and that the ingredients of the MD closely interact with gut microbial community, it can be suggested that the mechanisms by which MD drives health effects on the host might be mediated by an integrated interplay between the diet and the gut microbiome (Figure 1). Two main mechanisms underlying these positive effects of MD are (a) beneficial modulation of the gut microbiota (i.e., increased microbial diversity and production of beneficial metabolites like SCFAs), and (b) reduced metabolic endotoxemia by suppressing the growth of gram-negative bacteria and improving the gut barrier integrity by modulating tight junctions and mucus secretion67. Both of these mechanisms can help in reducing the systemic inflammation, which is the hallmark of many chronic diseases in the human body. Table 2 presents some of the reported beneficial health effects of Mediterranean-style diets and summarizes how different MD components could prevent/ameliorate these disorders by promoting a homeostatic gut microbiome and permeability to maintain the balanced arrays of the bacterial metabolites and the inflammatory molecules across the gut epithelium.\n\n\nMicrobiome-mediated pro-health effects of MD: purported and speculated mechanisms\n\nExtensive literature is available about the health benefits of MD; however, knowledge concerning the impact of MD on gut microbiota-mediated disease outcomes remain limited. The precise mechanism by which MD confers its beneficial effects on host health such as lowering the risk of cardiovascular, metabolic and gastrointestinal diseases and certain cancers remains unclear. However, most of these beneficial effects have long been speculated to be mediated by several interrelated and overlapping factors including cholesterol-lowering effect, protection against oxidative stress and inflammation, modification of hormones/growth factors involved in carcinogenesis, and control/inhibition of nutrient-sensing pathways by restriction of specific amino acids66. In addition, recent literature suggests that diet has a major impact on gut microbiome and the production of gut microbiota-derived microbial metabolites, which can considerably influence the host metabolic health68. Ever-mounting metagenomic studies have suggested that specific nutrients, particularly dietary fiber and proteins, exert strong effects on gut microbiota composition as well as on the production of microbial metabolites that further influence multiple features of the host immune and metabolic health69,70. For example, trimethylamine N-oxide (TMAO), a gut microbial metabolites from dietary choline and L-carnitine, is known to increase the risk of cardiovascular diseases independently of cardio-metabolic risk factors71. TMAO, at abnormally high levels, can induce vascular inflammation and prothrombosis by exaggerating platelet hyper-responsiveness to multiple agonists, and could be implicated in the pathogenesis of obesity and type 2 diabetes72,73. Given that a typical Mediterranean-style diet contains a proportion of choline and L-carnitine (which are abundant in eggs, red meat, and cheese) that is over 50% lower than a typical Western-style diet, it could be speculated that some of the beneficial effects of MD on host cardio-metabolic health might at least partly be mediated via such microbiome-related mechanisms. On the other hand, studies have shown that a poor adherence to a Mediterranean-style diet is associated with higher urinary TMAO level16. It is acknowledged that the physiological effects of the gut microbiota on host health are mediated not only by the direct interaction of the microbe with the host but also equivalently by the indirect microbial processes including the production of fermentation metabolites from diet or de novo. Thus, the array of microbial metabolites in the host gut from dietary fermentation is a function of not only the gut microbial ecology but also the substrate such as the fermentation of complex carbohydrates (e.g., fiber) or other plant-based foods, which are abundant in a Mediterranean-style diet, that leads to the production of beneficial SCFAs, in contrast to TMAO, which is frequently observed in people consuming Western-style diets poor in minimally processed plant-based foods and rich in processed red meats.\n\nMD is also rich in complex and insoluble fiber content when compared to a typical Western-style diet. A high intake of dietary fiber is well known to promote the beneficial modulation/maintenance of the gut microbiota with a reduced population of Firmicutes, while increasing that of Bacteroidetes, thereby yielding high levels of SCFAs including butyrate in the gut. These microbiota-derived metabolites including acetate, propionate and butyrate are known to protect against the development of several intestinal, inflammatory and allergic disease, and some of these effects are thought to be mediated via binding of these metabolites to specific G-protein-coupled receptors expressed on enteroendocrine and immune cells74. Adherence to a Mediterranean-style diet has been shown to reshape the gut microbiota of obese individuals, with increased population of Bacteroides, Prevotella, Roseburia, Ruminococcus, and Faecalibacterium prausnitzii, which are known for their fibrolytic activity, producing SCFAs by metabolizing carbohydrates18. Notably, Bacteroides fragilis and F. prausnitzii are also known to confer anti-inflammatory effects via inducing CD4+ T cells, the secretors of the anti-inflammatory interleukin-1075,76. In addition, the high content of vegetables, legumes, and fruit in a Mediterranean-style diet are also found to be associated with increased intestinal levels of short-chain fatty acids. These reports suggest that the Mediterranean-style diet fosters beneficial bacteria in the gut, which subsequently secretes beneficial metabolites.\n\nAnother reason that could underlie the health beneficial effects of Mediterranean-style diet is the lower intake of processed foods, as the Mediterranean dietary pattern is rich in minimally processed food, unlike a Western-style dietary pattern, which contains a much higher proportion of processed plant- and animal-based foods. This minimized intake of processed plant food with restricted calorie intake through MD is also known to confer positive effects on the gut microbiota diversity and composition by protecting/fostering the homeostasis of population levels of several beneficial bacterial groups77. On the other hand, long-term adherence to a Western-style dietary pattern, which has very low content of complex, insoluble, minimally processed, and microbiota-accessible plant-based fibers and carbohydrates, can lead to a dysbiotic gut microbiota, with various subdominant but important bacterial groups vanishing over generations, and may also negatively influence several important features of the host immune system, thereby increasing the predisposition to various gastrointestinal, metabolic and immune diseases74,76,78. Several studies have shown that the healthier and calorie-restricted dietary patterns with a high proportion of minimally processed plant-based foods can reprogram several important gut microbial functions that are crucial for promoting host health and wellbeing77,79.\n\nEmerging evidence shows that the adherence to a MD is associated with higher gut microbial diversity. The CORonary Diet Intervention with Olive Oil and Cardiovascular PREVention (CORDIOPREV) study involving 138 participants with metabolic syndrome and 101 healthy counterparts has reported restoration of the gut microbial dysbiosis in metabolic syndrome patients following long-term adherence to the MD, although the disease persisted80. Interestingly, the study demonstrated that the MD adherence could restore the patients’ gut microbiota in a similar way to the gut microbiota composition seen in metabolically healthy subjects, by fostering the population of saccharolytic bacterial genera, including Bacteroidetes, Faecalibacterium, Roseburia and Ruminococci, all of which are known to be associated with increased fermentation capacity to produce healthy SCFAs in the gut. A long-term adherence to the MD has also been found to increase the population of Roseburia sp. and Oscillospira sp., in addition to improving insulin sensitivity in obese people81. Notably, Roseburia is a prominent butyrate-producing genus that has been found to confer anti-inflammatory effects and is generally found to be reduced in type-2 diabetes patients82,83. These reports suggest that a MD might be effective in the prevention and management of type-2 diabetes, although more studies are needed to affirm these therapeutic effects in particular reference to their connection with gut microbiota-associated factors.\n\nAltogether, these reports suggest that the MD modulates gut microbiota profiles (such as higher population levels of Bacteroidetes, Clostridium cluster XIVa, Faecalibacterium prausnitzii, Lactobacilli, and Bifidobacteria, or lower Firmicutes) and can influence the diversity, activities and functionalities of various gut bacteria, thereby also fostering healthy metabolites (such as SFCAs) that can confer multiple benefits to the host intestinal, metabolic and immune health. Nevertheless, broader and more inclusive clinical and epidemiological studies assessing the temporal changes in the composition and function of gut microbiota enterotypes are still needed to establish a gut microbiome signature as a marker of MD adherence.\n\n\nNon-human primates (NHPs): an ideal model to elucidate diet-microbiome interactions in human health and disease\n\nGiven the profound impact of diet on the diversity and composition of host gastrointestinal microbiome68,84–86, the gut microbiome can be illustrated as a valuable biomarker of long-term intake of healthy or unhealthy diets. Therefore, it is imperative to elucidate whether, how and to what extent these long-term dietary patterns can influence the composition of the gut microbiota and how this could affect the production of beneficial microbial metabolites. As mentioned above, diet shapes the gut microbiome by supplying specific substrates that differentially foster the growth of specific gut microbial communities87–89; this diet-microbiome network is universally consistent in human and animal studies85,87,90,91. Specifically, the majority of diet-microbiome-targeted studies focus on high-fat, high-sugar, low-fiber vs. low-fat, low-sugar, high-fiber diets, with particular reference to nutrition- and gut-related maladies, including obesity, endotoxemia, insulin resistance, type-2 diabetes, and other metabolic syndromes92,93. Several animal models, including those in mice, hamsters, rats, guinea pigs and zebrafish are used for investigating diet-microbiome interactions, but, given the specific and prominent impact of dietary patterns on the gut microbiome, these models might not invariably be truly translatable to human milieus owing to far-reaching dissimilarities in their dietary regimen, age, body size and environmental elements. Accordingly, novel models such as NHPs are being sought for investigations of human diets and their interactions with gut microbiome, to decipher better understanding in human inference92,94–100. In addition, while it is challenging to precisely control and monitor dietary patterns through questionnaires in human interventions, studies performed on small animals are disadvantaged by short-term diet intervention periods. To overcome these limitations, NHPs represent an excellent model for investigating the diet-microbiome interactions and their impact on host health. Their physiological and phylogenetic closeness to humans makes them a perfect and biologically relevant animal model for human context to examine diet-microbiome connections as well as for studying the relation of this network with different nutrition- and gut-related diseases95,96,99–102. In one such endeavor, we recently performed a study wherein we demonstrated the effect of a long-term Western- vs. Mediterranean-style diet intake on the gut microbiome composition in NHPs (Cynomolgus monkeys; Macaca fascicularis)21. Previous reports have demonstrated that the gut microbiome of NHPs resembles more closely to those of primates than other animals103. The human gut largely harbors microbes belonging to nine different bacterial divisions, viz. Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, Verrucomicrobia, Fusobacteria, Spirochaetes, Cyanobacteria, and VadinBE97104,105. Interestingly, our NHP data also demonstrated Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fibrobacteres, Spirochaetes, Cyanobacteria, and Tenericutes as most abundant bacterial phyla21. This again validates that the NHP microbiome studies can yield important indications about specific features of these bacterial clades in the human gut and provide unique opportunities to investigate diet-microbiome interactions.\n\n\nEffect of MD on NHP microbiome: similar findings as seen in human and rodent studies\n\nOur data obtained feeding a MD to NHPs also demonstrated that MD boosted the gut microbiome diversity and promoted the carriage of several important bacterial groups, including Bacteriodes, Prevotella, Lactobacillus, Faecalibacterium, Clostridium and Oscillospira21, again corroborating that the host diet can influence gut microbiome diversity and composition. Notably, similar effects as seen in the NHP cohort have also previously been seen in human studies (Figure 2). For example, diets rich in fiber and unsaturated fatty acids are known to help maintaining a healthy and diverse gut microbiome. Our NHP data also demonstrated higher alpha-diversity in NHPs consuming the MD, an effect that has also been observed in several other studies on humans or animal animals68,78,99,106 and can be attributed to a higher proportion of fiber in the MD. In addition, our NHP study also demonstrated higher Bacteroidetes-Firmicutes ratio and higher abundance of Clostridium and Prevotella in NHPs on MD. Similar results have been reported by previous studies performed in humans and other mammals reporting that MD (and specifically the higher fiber intake and less intake of high-glycemic index sugars) positively alters the gut microbiota, with an increased Bacteroides, Clostridium and Prevotella population8,88,99,107–109 whereas a low-fiber, high-fat and high-sugar diet is linked with increased Firmicutes and reduced Bacteroides population88. Another interesting observation was the increased abundance of genus Oscillospira in MD-fed NHPs. Oscillospira, a genus from the Ruminococcaceae family, is usually found prevalent in the gastrointestinal tract of ruminants consuming diets rich in complex plant fibers and hence is regarded as genus adapted to vegetable-rich diets, such as Mediterranean-style diets110,111.\n\nAnother interesting observation from this NHP study was the increased abundance of Faecalibacterium prausnitzii, one of the beneficial butyrate-producer previously reported to be found in higher numbers in humans consuming a Mediterranean-style diet111–113. Various human and small animal studies have reproducibly reported fostering of a population of beneficial bacterial groups, including Lactobacillus sp114–116. and Faecalibacterium sp117. following consumption of diets that are rich in complex carbohydrates, such as prebiotics. In addition, omega-3 fatty acids, which are present in higher ratio in Mediterranean-style diet, can also stimulate the intestinal population of several beneficial bacteria including lactobacilli that typically inhabit the distal gut, a primary site for the metabolism of mono- and poly-unsaturated fatty acids9,118,119. Interestingly, in a recent study on these NHPs, we demonstrated that the consumption of MD also leads to an increased Lactobacillus abundance and increased bacterially processed bioactive compounds in the mammary glands compared with Western diet-fed monkeys, clearly indicating that the influence of MD on the microbiome reaches far outside the gut in distal sites such as the mammary glands and could also establish an alternative pathway for breast cancer prevention120. Conversely, high-fat diets are also known to reduce the intestinal carriage of lactobacilli93,121,122. In addition, we have recently reported that MD also protects these NHPs against hepatosteatosis while reducing BMI and body fat123. Hence, these common findings between NHP and human studies clearly demonstrate and advocate that NHPs can prove to be an excellent experimental animal model to examine the diet-microbiome interaction in context to human health and disease.\n\nAltogether, these findings from our cohort of the MD-fed NHPs demonstrate that the positive modulation of the gut microbiome is concomitant with the beneficial effects of MD on host health and hint that this microbiome modulation might even at least in part also mediate/underlie the health effects of MD, particularly given that the majority of the MD-induced changes in the gut microbiome including enhanced microbial diversity and fostering of several beneficial bacterial groups are known to be beneficial for host intestinal, metabolic and overall health21. However, although the health benefits of MD have been studied extensively, research on MD’s impact on microbiome-mediated disease outcomes still remains undetermined. For instance, it is known that MD consumption leads to gut microbiome alterations; however, the magnitude to which these alterations occur may be potentially impacted by multiple factors such as the study duration, host age and lifestyle habits, specific disease predisposition or severity, level of dietary adherence, etc. which otherwise remain to be explicated and hence would be prerequisite to pinpoint the factors contributing to the outcome of MD on gut microbiome and deliver conclusive data on the role of gut microbiome in MD’s beneficial health outcomes124,125. As a result, according to the existing literature, the examination of gut microbial composition could not be endorsed as a stand-alone tool for the health effects of MD. To this end, systemic approaches that incorporate gut metagenomics, transcriptomics and metabolomics could help elucidate the complex association of MD-microbiome interrelationship with host health.\n\n\nPerspectives on the future\n\nRecent evidences from animal and human studies are beginning to elucidate the mechanisms underlying the pro-health effects of the traditional MD. The metabolism and fermentation of plant-based foods packed with complex fibers, a wide variety of vitamins and phytochemicals, could also play an important role in promoting host metabolic health. Although more research is needed, specific dietary patterns (particularly, the Mediterranean-style diet) have been found to have a strong influence on the gut microbiome composition and metabolic function, suggesting that there is an opportunity to prevent and treat various lifestyle-related disorders based on gut microbiota outcomes. Given the plasticity of the gut microbiota, it can be speculated that such Mediterranean-style nutritional intervention can positively affect host health, either by maintaining a physiologically homeostatic gut microbiota configuration or by reversing the gut dysbiosis state; this positive modulation of the gut microbiome could be considered as a potential and holistic target for non-pharmacological interventions in a variety of disease states. The potentiality of the gut microbiome is tremendous. It can be easily and rapidly modulated in a natural, non-invasive, non-pharmacologic way, i.e., via diets and other dietary supplements like probiotics and prebiotics. The ever-mounting knowledge of the gut microbiome as acquired by the use of remarkable high-throughput sequencing and omics tools in combination with the data from various in vitro and in vivo experimental models is shedding a new light on the key mechanisms through which diet-microbiome crosstalk in the gut can modulate potential risk factors of several human diseases. This wealth of knowledge is certainly going to offer potential avenues for decoding the complexities and functionalities of the diet-microbiome interface and for developing personalized dietary strategies to determine the healthiest dietary pattern for the personalized host.\n\nIn conjunction with gut microbial composition in response to diets like MD and the Western diet, it is becoming evident that the microbial metabolites produced by gut microbiome and dietary interactions plays an important role to regulate host metabolism and physiology126. MD feeding enhances abundance of probiotics or beneficial bacteria like lactobacilli21, however, future studies focusing on determining how the increased abundance of lactobacilli benefit host health will be important to conclude the mechanistic views. It is difficult to pinpoint exact molecular mechanism(s) by which diets and ingredients can impact human health; however, several layers of mechanistic studies like systemic, organ- and cell-based mechanism should be the focus of future research. Such studies can provide important information to include the vital ingredients that not only enhance the impact of diets like MD, but can easily be tailored for personalized nutritional approaches. Combining gut microbiome, and metabolic response (like the glycemic index) of diets on individual basis are attractive area of current research, that can extend valuable outcome in the field of diet-microbiome interactions to benefit human health.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Grant information\n\nThe authors gratefully acknowledge funding support for this work from the Center for Diabetes, Obesity and Metabolism, the National Institutes of Health funded the Wake Forest Alzheimer's Disease Research Center (funded by P30AG049638); OAIC Pepper Center (P30AG021332); R01AG058829; R01DK099164; R01AG018915; R01HL122393; R01HL087103 and the Clinical and Translational Science Center (Clinical Research Unit, funded by UL1TR001420), all at Wake Forest School of Medicine. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLecomte V, Kaakoush NO, Maloney CA, et al.: Changes in gut microbiota in rats fed a high fat diet correlate with obesity-associated metabolic parameters. PLoS One. 2015; 10(5): e0126931. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShively CA, Appt SE, Vitolins MZ, et al.: Mediterranean versus Western Diet Effects on Caloric Intake, Obesity, Metabolism, and Hepatosteatosis in Nonhuman Primates. Obesity (Silver Spring). 2019; 27(5): 777–784. PubMed Abstract \| Publisher Full Text\n\nCani PD: Human gut microbiome: hopes, threats and promises. Gut. 2018; 67(9): 1716–1725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGutiérrez-Díaz I, Fernández-Navarro T, Sánchez B, et al.: Mediterranean diet and faecal microbiota: a transversal study. Food Funct. 2016; 7(5): 2347–2356. PubMed Abstract \| Publisher Full Text\n\nYadav H, Jain S, Bissi L, et al.: Gut microbiome derived metabolites to regulate energy homeostasis: how microbiome talks to host. Metabolomics. 2016; 6: e150. Publisher Full Text" }	[ { "id": "52604", "date": "15 Oct 2019", "name": "Prakash Motiram Halami", "expertise": [ "Reviewer Expertise Probiotics", "gut microbiota", "antibiotic resistance in lactic acid bacteria & antimicrobial peptides" ], "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 have summarized the importance of consuming a Mediterranean-style diet, which is associated with prevention of metabolic and cardiovascular diseases. It is know that a Mediterranean-style diet (MD) contributes to the high proportion of fibre, mono- and poly-unsaturated fatty acids, antioxidants and polyphenols. The authors have also stated the beneficial modulation of the gut microbiome is associated with MD as evident from in-vivo experimental studies. The consumption of MD has provided many health benefits which is associated with rich microbial diversity, composition and its function. In this review article, the author have discussed, with evidence, the MD in rodent & human subjects, as well as the importance of gut bacteria and microbial metabolites. The article has been well-written with reference to bile, gut microbiota & chronic diseases, where in authors have underlined the benefits of MD. They have also summarised western style of dietary habits associated with chronic diseases. Further, the authors have provided information on MD, its intangible culture and nutritional dietary pattern along with the ingredients generally used. In subsequent sections, MD and its positive health effects and possible mechanisms is provided in a tabulated form. The authors have presented a diagrammatic overview of the consumption of MD and its effect on gut microbiota, especially on microbiome diversity by minimizing the occurrence of firmicutes and proteobacteria. In subsequent sections the authors have mentioned the microbial mediated beneficial effects of MD and it possible mechanisms. The authors have mentioned the use of Non-human Primates (NHP) for studying diet microbial interaction with reference to human health and diseases. In subsequent sections, the authors have mentioned the effects of MD on the microbial diversity of NHP and its correlation with human subjects, distinct microflora associated with human subjects e.g. Ruminococcus is also described. In the 'Perspectives on the future' section the authors have highlighted the importance of plant based foods and its importance to host metabolic rate etc. The microbiome composition and metabolite production that are required to prevent various lifestyle disorders have been suggested. Authors have also provided information on how probiotics can influence the gut microbiome of the personalized hot. Accordingly, authors have prepared the article well and following comments are suggested:\n\nComments:\nThe use of the acronym MD for \"Mediterranean-style diet\" should be consistent throughout the MS.\n\nIn table 2, please change \"SCFA\" in possible mechanism to \"SCFA production\".\n\nThe many repeated MD ingredients mentioned in table 2 could be better represented by combining a few of the rows.\n\nIn figure 1, it is suggested that the font colour for the increased parameters could be green and the those for decreased one could be red.\n\nAuthors could include a section for the role of prebiotic compounds that are found in MD.\n\nA concern when comparing NHP microbiome with that of the human microbiome is that is all the interaction (enzymatic or hormonal) similar to that of NHP? As any difference in these factors in the two species can result in misleading conclusions.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "65835", "date": "13 Jul 2020", "name": "Rajeev Kapila", "expertise": [ "Reviewer Expertise Health validation of nutraceuticals (Probiotics and Bioactive peptides) and their mode of actions" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the current perspective, the authors have lucidly briefed that shifting of dietary-pattern from ‘Western-style’ dietary habits to a ‘Mediterranean-style‘diet effectively target gut microbiome interactions by diversifying and increasing the richness of gut microbiome which can justifiably help in prevention and treatment of various chronic diseases like obesity, diabetes, cancer and aging-related diseases such as Alzheimer’s disease. In a quintessential and step-by-step manner, the authors have well described the usefulness of the Mediterranean-style diet which represents a promising, efficacious, and holistic approach to restore host heath.\n\nIn this article, the authors have discussed the nutritional characteristics of a typical Mediterranean-style dietary pattern along with their components having an association with various health benefits. Briefly discussed the adverse effects of TMAO released from Western-style diet over beneficial effects of SCFA released during dietary fermentation of complex carbohydrates present in Mediterranean-diet. Thus authors have also suggested some underlying mechanisms by which the purported health benefits of MD can be exerted. The authors have justified the use of non-human primates as an ideal model to dissect diet-microbiome interactions in human health and disease after drawing evidence from the fact that the diet-microbiome network is universally consistent in human and animal studies. In a nutshell, the manuscript is nicely written with sufficient facts and figures and the following comments are suggested for each section of the manuscript wherever they may apply\nComments:\nIt could be better if a tabular description of Western-style’ dietary habits and their components would also be made highlighting the difference in components.\n\nRelease of metabolites such as SCFA is now well recognised as epigenetic modifiers which could be an alternative underlying mechanism of beneficial effects of Mediterranean-style dietary pattern due to the abundance of prebiotic and polyphenolic components which may also be highlighted in proposed biochemical pathways.\n\nWriting “Mediterranean dietary pattern” as rich in minimally processed or unprocessed food, unlike a Western-style dietary pattern appears faulty because cooking and frying used in these dietary patterns are also a form of food processing hence it is suggested to use different terminology to differentiate between processed / un-processed/ cooked foods.\n\nIn the introduction section sentence “mono- and poly-unsaturated fatty acids such as fish and olive oil” could be written as mono- and poly-unsaturated fatty acids consumed through fish and olive oil.\n\nUnder the heading “Mediterranean diet: the intangible cultural and nutritional dietary pattern” the sentence “A Mediterranean-style diet typifies a nutritionally balanced diet, characterized by intake in high amounts and frequency of important sources of fibers” may be modified as “A Mediterranean-style diet typifies a nutritionally balanced diet, characterized by high and frequent intake of important sources of fibres”.\n\nIt would be better if some references are cited with “Table 1”, if available.\n\nIn Table 2, Figure 1 and 2, use different yet consistent colours for ↓↑ respectively.\n\nUnder the heading “Microbiome-mediated pro-health effects of MD: purported and speculated mechanisms” the sentence “Thus, the array of microbial metabolites in the host gut from dietary fermentation…... is too long hence better to modify for more clarity.\n\nSimilarly under the heading “Effect of MD on NHP microbiome: similar findings as seen in human and rodent studies” the sentence “Altogether, these findings from our cohort of the MD-fed NHPs demonstrate that the positive modulation of the gut microbiome....... is very lengthy may be re-written for better understanding\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": "65807", "date": "21 Aug 2020", "name": "Raghvendra Kumar Mishra", "expertise": [ "Reviewer Expertise 1. Arachidonic acid production and molecular characterization of Mortierella alipna isolates (Filed 02 patents", "01 paper", "01 submitted). 2. Biosynthesis of Nanoparticles from C. rosesus (01 paper published in Frontiers in Microbiology other in pipe line). 3. Development of EST-SSR and ILP data for commercially important plan variety. (03 papers published", "one project is going on)" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes very timely and important review on how diet and microbiome interactions play a crucial role in maintaining health, especially mental health. This manuscript has been well written, cited all important references properly, and easy to read with nice graphic. I strongly believe that indexing this manuscript on time will provide high readership to the journal.\nI find this topic very interesting with well written paper, thus recommend indexing of this interesting manuscript immediately.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-699
https://f1000research.com/articles/8-150/v1	04 Feb 19	{ "type": "Review", "title": "Investigating colistin drug resistance: The role of high-throughput sequencing and bioinformatics", "authors": [ "Dickson Aruhomukama", "Ivan Sserwadda", "Gerald Mboowa", "Ivan Sserwadda", "Gerald Mboowa" ], "abstract": "Bacterial infections involving antibiotic resistant gram-negative bacteria continue to increase and represent a major global public health concern. Resistance to antibiotics in these bacteria is mediated by chromosomal and/or acquired resistance mechanisms, these give rise to multi-drug resistant (MDR) or extensive drug resistant (XDR) bacterial strains. Most recently, a novel acquired plasmid mediated resistance mechanism to colistin, an antibiotic that had been set apart as the last resort antibiotic in the treatment of infections involving MDR and XDR gram-negative bacteria, has been reported. Plasmid mediated colistin resistant gram-negative bacteria have been described to be pan-drug resistant, implying a state devoid of alternative antibiotic therapeutic options. This review describes the evolution of antibiotic resistance to plasmid mediated colistin resistance, and discusses the potential role of high-throughput sequencing technologies, genomics and bioinformatics towards improving antibiotic resistance surveillance, the search for novel drug targets and precision antibiotic therapy focused at combating colistin resistance, and antimicrobial resistance as a whole.", "keywords": [ "Antibiotic resistance", "Colistin resistance", "Pan-drug resistance", "Gram negative bacteria", "Genomics", "Bioinformatics" ], "content": "Introduction\n\nIn the recent past, old antibiotic classes previously deemed unfit for treatment of bacterial infections due to associated toxicity concerns have been recommended for treatment in this type of infection1,2. This has been attributed to the emergence of resistance to the most recently considered last line antibiotics, the carbapenems1,2. Carbapenem resistance has been documented in bacteria belonging to the Enterobacteriaceae family, Acinetobacter baumannii and Pseudomonas aeruginosa1,2. The adoption of the old antibiotic agent category in routine empirical treatment has witnessed the use of a number of antibiotics such as colistin1,2.\n\nDespite this reversion, gram-negative bacteria continue to undergo chromosomal mutations, which render their respective treatments virtually impossible and hence a major threat to global public health. The effects of these antibiotic resistance mutations are further exacerbated by horizontal transfer of antibiotic resistance genes in the same bacteria. As such, this paper explores the current documented trends of colistin resistance in several African settings. Additionally, it also describes the evolution of antibiotic resistance to plasmid mediated colistin resistance and the potential role of genomics and bioinformatics in precision antibiotic therapy targeted towards combating colistin resistance and antimicrobial resistance.\n\n\nColistin resistance trends in Africa\n\nData on the antimicrobial resistance burden, particularly colistin resistance, in Africa remains limited3. In 2014, the World Health Organization reported that antimicrobial resistance surveillance in Africa was a particularly difficult feat due to the scarcity of viable medical data, statistical information and unreliable laboratory capacity3. Despite this, African countries remain un-exempted from this worldwide antibiotic resistance trend that has emerged not only within hospital settings, but also disseminated within the community. The limited available literature from African settings has reported the mcr-1 gene mediated colistin resistance to be most prevalent in Africa, largely in South Africa, and this covers the largest portion of Africa according to the global map (Figure 1)4–7. Beyond South Africa, the emergence of colistin resistance has been reported in Algeria, Rwanda and Uganda8–10.\n\nAdapted from Xavier et al.4 under a CC-BY 4.0 license.\n\n\nEvolution to plasmid mediated colistin resistance in gram-negative bacteria\n\nColistin (polymyxin E) is part of an old generation of antibiotics11 that form a family of cationic polypeptides. These are characterised by having a lipophilic fatty acyl side chain12–14. No exact mechanism of bacterial killing has been documented for polymyxins, especially in Acinetobacter spp14,15. However, a two-step mechanism has been described to elucidate their possible mechanism of action13,14.\n\nThe two stages involve: i) initial binding to and permeabilization of the outer membrane and ii) the destabilisation of the cytoplasmic membrane of the bacteria12–14. As a consequence, colistin functions by intercalating into the inner membrane following diffusion from the outer membrane across the periplasm and consequently causing the formation of pores, a phenomenon that results in bacterial lysis, which follows initial binding to bacterial surfaces12–14. Initial binding of colistin to the bacterial surface chiefly depends on the electrostatic interaction between the positively-charged colistin and the negatively charged phosphate group of lipid A, an endo toxic component on the lipopolysaccharide localised on the outer leaflet of the bacterial outer membrane12,14.\n\nThe modifications of the lipid A, which reduce and/or abolish the initial charge-based interaction with the polymyxins in bacteria13,15,16 and also the addition of either/or the 4-amino-4-deoxy-L-arabinose (L-Ara4N) and the phosphoethanolamine (PEtn) that ultimately form the basis of colistin resistance in bacteria16, is mediated by chromosomally encoded genes. These are involved in the modulation of two component regulatory systems; PmrA/PmrB and PhoP/PhoQ and mgrB, a negative regulator of the PhoP/PhoQ signalling system13–16.\n\nAlthough initially thought that this resistance could not be spread from cell to cell (plasmid mediated)16, currently studies have shown otherwise. These have alluded transfer of colistin resistance among bacteria via plasmids in horizontal gene transfer4,16,17. Plasmid transfer of the colistin resistance mobile genes, mcr-1, mcr-2, and also mcr-34,16.\n\n\nPan-drug resistance and characteristics of colistin resistant gram-negative bacteria\n\nThe treatment of infections involving antibiotic resistant gram-negative bacteria has become increasingly difficult overtime, a factor that has greatly contributed to high morbidity, mortality and high costs of health care18,19.\n\nCurrently, antibiotic resistance in these bacteria spans across several classes but likely follows a precise hierarchy of acquisition; this is mostly characterised by acquisition of “enhanced resistance” against more potent antibiotics following primary acquisition of “weaker resistance” against the less potent antibiotics alongside intrinsic resistance mechanisms in these bacteria, a trend that follows a Darwin’s like fashion20–23. These changes are a function of horizontal gene transfer, via conjugation, transformation and transduction24–27.\n\nResistance in gram-negative bacteria has been seen to transit from being mediated by the extended spectrum β-lactamases, a group of enzymes that can be disseminated among bacteria28,29; these chiefly confer resistance against broad spectrum cephalosporins. However, they also confer resistance to penicillins, monobactams and some carbapenems, particularly the Klebsiella pnemoniae carbapenemase, KPC28,30,31. In the same hierarchy are AmpC β-lactamases that form another group of β-lactamases, derived from older broad spectrum β-lactamases. These provide an even more extended activity that includes resistance against the cephamycins alongside resistance to penicillins, monobactams and cephalosporins32–34. These enzymes have in recent times been shown to not only be limited to being encoded on the chromosomes of bacteria, but have also been documented to have the potential of being disseminated via plasmids in horizontal gene transfer28,34,35 and also to co-exist with the extended spectrum β-lactamases36,37; factors that have made these bacteria “better resistant” to antibiotics. Next in the hierarchy are the carbapenemases, these enzymes are chiefly acquired in horizontal gene transfer and confer resistance to carbapenems alongside resistance to penicillins, broad spectrum cephalosporins including cefepime, a fourth generation cephalosporin, monobactams, aminoglycosides, quinolones and fluoroquinolones28,38. The development of resistance mediated by these enzymes to the different classes of antibiotics in these bacteria has been attributed to various factors among which is their use in therapy. This has not only abetted maintenance of resistance via selecting for resistance to these antibiotics in these bacteria but has also created a gap, a need for alternative antibiotics in therapy to replace the penicillins, β-lactams, carbapenems and the other classes of antibiotics used in the treatment of infections involving the drug resistant gram-negative bacteria4,16.\n\nColistin, a polypeptide antibiotic, a relatively old antibiotic, has been currently relied upon to provide the ultimate line of refuge against infections caused by antibiotic resistant gram-negative bacteria despite its previously documented impacts on health4,16. Colistin also appears to offer a choice in the face of almost no new antibiotics in production pipelines4,16,17.\n\nWorryingly, the use of colistin is under threat due to the development of the novel plasmid mediated colistin resistance mediated by mcr-1, mcr-2 and mcr-3 heralds4,16. This provides a new challenge as bacteria that express these resistance genes assume the lead in the antibiotic resistance hierarchy and are distinctively extensive or worse pan drug resistant16,39–42.\n\nMolecular studies previously done have reported colistin resistant gram-negative bacteria to also be resistant to an array of antibiotics. These bacteria have also been reported to carry plasmids that have been found to carry alongside colistin resistance genes, β-lactamases43,44, carbapenemase encoding genes45 and genes that code for resistances to other antibiotic classes that may include quinolones, fluoroquinolones and aminoglycosides13. Additionally, the carriage of mcr-1 has been documented as a possible indicator of resistance to the third generation cephalosporins and carbapenems38,44. Furthermore, these genes have been found to be co–carried with other resistance determinants in plasmids13,44,46,47; these genes represent a novel mechanism of antibiotic resistance in bacteria and a threat to the existing antibiotic therapy. Worsening the situation is the ability of selection for colistin resistance via the use of the extended spectrum cephalosporins. Additionally the use of tetracycline and sulphonamides has also been reported to contribute to the dissemination of colistin mobile gene carrying plasmids44,46. Also, worth noting is plasmids that carry colistin resistance genes have also been found to mostly carry other antibiotic resistant genes13,44–46.\n\n\nThe role of high-throughput sequencing technologies and bioinformatics\n\nAdvances in technology including the rapidly growing field of genomics, are transforming clinical medicine48 and high-throughput sequencing technology (HTS) is increasingly being used in clinical microbiology49. HTS, with relatively simple bench top technology and efficient genomic library preparation protocols, has significantly improved the capacity to perform low-cost, efficient whole-genome sequencing (WGS), and has made it a feasible tool to enhance clinical diagnostic investigations in near real-time48. The processes generally involve culture-free parallel sequencing, producing vast quantities of genomic data that require modern computation techniques to assemble the genomic sequence reads as well as performing ensuing analyses that range from identifying the bacterial species or strain, antibiotic resistance mutations in the bacterial genomes, while ensuring the highest possible discriminatory power ever achieved by any technology49. Apart from this, WGS of bacteria can identify genes associated with virulence and pathogenicity as well as discover new genetic mechanisms for virulence, pathogenicity and antibiotic resistance48,50,51.\n\nThe identification and prediction of antibiotic resistant microorganisms in clinical specimens solely by molecular means in the diagnostic microbiology laboratory is not novel52. HTS technologies and computational tools offer unprecedented ability to sequence multitudes of bacterial genomes and enable interpretation of the resultant sequence information in near “real-time”52.\n\nWGS represents the pinnacle for bacterial strain characterisation and epidemiological analyses. It is rapidly replacing traditional typing methods, antibiotic resistance gene detection and other molecular-based investigations in the near future. HTS technologies are rapidly evolving and their implementation in clinical and public health microbiology laboratories is increasing at a similar pace. These require standardised sample quality control, data interpretation, bioinformatics expertise, and infrastructure. The term ‘bioinformatics’ encompasses the handling and analysis of genomic sequence data, usually with the assistance of computer-based algorithms. Both ‘open source’ and commercially available bioinformatics programs/tools have been specifically developed for use in a clinical setting. However, many of practising healthcare workers in current practice have limited bioinformatics knowledge48.\n\nFurthermore, phenomena such as genome plasticity and pan genomes that have the ability to influence bacterial resistome can only effectively be investigated using HTS and bioinformatics analyses. Understanding the bacterial genome dynamics is an important step in identifying the forces behind the observed antibiotic resistance and therefore be able to effectively manage the disease in question.\n\nThe bottleneck that remains in implementing WGS for clinical purposes is post-sequencing data analysis48.\n\n\nFuture direction of HTS\n\nAntibiotic resistance in bacteria is generally a natural phenomenon53–55 though augmented by human behaviour. Therefore, it is imperative to harness the best HTS technologies that sequence DNA at unprecedented speed, to enable previously unimaginable scientific achievements and novel biological applications56. Such applications of genomics tools has revolutionized microbial ecological studies and drastically expanded our view on the previously underappreciated microbial world57 including acquisition and transmission dynamics of antibiotic resistance. Single-Molecule Real-Time (SMRT) sequencing (Pacific Biosciences Inc.) in clinical microbiology has finally been realized at many levels in health care systems in the developing world and relatively only used during isolated scenarios of disease outbreaks in the less developed countries. These developments in HTS must be matched with continued efforts to improve the current bioinformatics analytic pipelines. Applying SMRT while genome sequencing to investigate bacterial colistin resistance would be made possible to predict resistance mutations, resistance mechanisms, trends, and patterns enabling efficient management of the colistin resistance by healthcare providers and pharmaceutical companies.\n\n\nConclusions\n\nIt is known that host, bacterial and environmental factors interact collectively to bring about antibiotic resistance. Therefore, HTS should be applied to a wide range of global collections of bacterial whole genomes to identify and predict new antibiotic drug resistance mutations using appropriate computational and bioinformatics algorithms.\n\nComputational algorithms and tools offer ability to simulate bacterial genomic mutations while also offering possible clues on the mechanisms that may be shaping these mutations. These can as well be utilised to develop therapeutic interventions that may be used to target both the current and future acquired antibiotic drug resistance mutations.\n\n\nData availability\n\nNo data are associated with this article", "appendix": "Grant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nGiske CG: Contemporary resistance trends and mechanisms for the old antibiotics colistin, temocillin, fosfomycin, mecillinam and nitrofurantoin. Clin Microbiol Infect. 2015; 21(10): 899–905. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohamudha PR, Harish BN, Parija SC: Molecular description of plasmid-mediated AmpC β-lactamases among nosocomial isolates of Escherichia coli & Klebsiella pneumoniae from six different hospitals in India. Indian J Med Res. 2012; 135(1): 114–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhosh B, Mukherjee M: Emergence of co-production of plasmid-mediated AmpC beta-lactamase and ESBL in cefoxitin-resistant uropathogenic Escherichia coli. Eur J Clin Microbiol Infect Dis. 2016; 35(9): 1449–1454. PubMed Abstract \| Publisher Full Text\n\nAnusuya Devi D, Ramachander Rajesh: Detection of Extended-spectrum Beta-lactamases in AMPC Co-producing Bacteria by Disc Diffusion Method in Clinical Isolates of Enterobacteriacae. Indian J Public Health Res Dev. 2016; 7(2): 236–242. Reference Source\n\nCapone A, Giannella M, Fortini D, et al.: High rate of colistin resistance among patients with carbapenem-resistant Klebsiella pneumoniae infection accounts for an excess of mortality. Clin Microbiol Infect. 2013; 19(1): E23–E30. PubMed Abstract \| Publisher Full Text\n\nAh YM, Kim AJ, Lee JY: Colistin resistance in Klebsiella pneumoniae. Int J Antimicrob Agents. 2014; 44(1): 8–15. PubMed Abstract \| Publisher Full Text\n\nOlaitan AO, Diene SM, Kempf M, et al.:Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study. Int J Antimicrob Agents. 2014; 44(6): 500–507. PubMed Abstract \| Publisher Full Text\n\nQureshi ZA, Hittle LE, O'Hara JA, et al.: Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin Infect Dis. 2015; 60(9): 1295–1303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGann P, Snesrud E, Maybank R, et al.: Escherichia coli harboring mcr-1 and blaCTX-M on a novel IncF plasmid: first report of mcr-1 in the United States. Antimicrob Agents Chemother. 2016; 60(7): 4420–4421. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFalgenhauer L, Waezsada SE, Yao Y, et al.: Colistin resistance gene mcr-1 in extended-spectrum β-lactamase-producing and carbapenemase-producing Gram-negative bacteria in Germany. Lancet Infect Dis. 2016; 16(3): 282–283. PubMed Abstract \| Publisher Full Text\n\nMalhotra-Kumar S, Xavier BB, Das AJ, et al.: Colistin resistance gene mcr-1 harboured on a multidrug resistant plasmid. Lancet Infect Dis. 2016; 16(3): 283–284. PubMed Abstract \| Publisher Full Text\n\nPoirel L, Kieffer N, Liassine N, et al.: Plasmid-mediated carbapenem and colistin resistance in a clinical isolate of Escherichia coli. Lancet Infect Dis. 2016; 16(3): 281. PubMed Abstract \| Publisher Full Text\n\nDu H, Chen L, Tang YW, et al.: Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant Enterobacteriaceae. Lancet Infect Dis. 2016; 16(3): 287–288. PubMed Abstract \| Publisher Full Text\n\nMonaco M, Giani T, Raffone M, et al.: Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014. Euro Surveill. 2014; 19(42): pii: 20939. PubMed Abstract \| Publisher Full Text\n\nKwong JC, McCallum N, Sintchenko V, et al.: Whole genome sequencing in clinical and public health microbiology. Pathology. 2015; 47(3): 199–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossen JWA, Friedrich AW, Moran-Gilad J: Practical issues in implementing whole-genome-sequencing in routine diagnostic microbiology. Clin Microbiol Infect. 2018; 24(4): 355–360. PubMed Abstract \| Publisher Full Text\n\nNijhuis RH, Oueslati S, Zhou K, et al.: OXY-2-15, a novel variant showing increased ceftazidime hydrolytic activity. J Antimicrob Chemother. 2015; 70(5): 1429–1433. PubMed Abstract \| Publisher Full Text\n\nFerdous M, Kooistra-Smid AM, Zhou K, et al.: Virulence, Antimicrobial Resistance Properties and Phylogenetic Background of Non-H7 Enteropathogenic Escherichia coli O157. Front Microbiol. 2016; 7: 1540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDunne WM Jr, Westblade LF, Ford B: Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory. Eur J Clin Microbiol Infect Dis. 2012; 31(8): 1719–1726. PubMed Abstract \| Publisher Full Text\n\nChandra H, Bishnoi P, Yadav A, et al.: Antimicrobial Resistance and the Alternative Resources with Special Emphasis on Plant-Based Antimicrobials-A Review. Plants (Basel). 2017; 6(2): pii: E16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWright GD: Q&A: Antibiotic resistance: where does it come from and what can we do about it? BMC Biol. 2010; 8(1): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrestinaci F, Pezzotti P, Pantosti A: Antimicrobial resistance: a global multifaceted phenomenon. Pathog Glob Health. 2015; 109(7): 309–318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J, Chiodini R, Badr A, et al.: The impact of next-generation sequencing on genomics. J Genet Genomics. 2011; 38(3): 95–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu J: Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances. Mol Ecol. 2006; 15(7): 1713–1731. PubMed Abstract \| Publisher Full Text" }	[ { "id": "45068", "date": "27 Mar 2019", "name": "Alexandre Angers-Loustau", "expertise": [ "Reviewer Expertise Molecular 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 this review, the authors describe the biology behind the evolution of antimicrobial resistance, with a focus on the resistance against colistin, one of the \"last-resort\" antibiotics. An alarming development was reported in 2016, where a form of resistance was discovered that could be readily transferred to other bacteria. Researchers first discovered this resistance in China, quickly followed by findings of similar resistance patterns in other countries. These discoveries relied in part on the use of high throughput sequencing.\n\nThe review is very short and it is understandable that, within these four pages, the authors can't expand these themes in too many details. Still, I have a few minor and a few major comments about the current version of the article.\n\nMinor comments:\nThe flow of the different sections of the review is a bit off in my opinion:\n\na) The section \"Colistin resistance trends in Africa\" describes the observed burden of colistin resistance in this continent\n\nb) the section \"Evolution to plasmid mediated colistin resistance …\" describes the biology and mechanism of colistin resistance, from chromosomal to plasmid-mediated.\n\nc) the section \"Pan-drug resistance and characteristics…\" describes, generally, the phenomena that gives rise to AMR. It would make more sense, to me, to rework these sections in the order c), b) then a).\n\nThe sentence \"Furthermore, phenomena such as genome plasticity and pan genomes that have the ability to influence bacterial resistome can only effectively be investigated using HTS and bioinformatics analyses.\" needs to be better explained and referenced. Also, pan genomes are not, in my opinion, \"phenomena\". The section \"Evolution to plasmid mediated colistin resistance...\" explains the mechanisms of action of chromosome-mediated resistance, then simply lists the genes involved in plasmid-mediated resistance, with no indication about modes of action of these genes, and whether it is different from the ones described previously. I noticed at least one incomplete sentence (\"Plasmid transfer of the colistin resistance mobile genes, mcr-1, mcr-2, and also mcr-3.\")\n\nMajor comments:\nThe section on Africa needs to be reworked. First, the Figure says \"Adapted from Xavier et al. under a CC-BY 4.0 license\". I don't know about the license and rights, but I didn't notice any difference with the original figure, so a more appropriate phrasing would be \"Reproduced from…\" (or, if I missed it, a better indication on how it was adapted). Second, I don't see how the figure supports the statement \"The limited available literature from African settings has reported the mcr-1 gene mediated colistin resistance to be most prevalent in Africa, largely in South Africa, and this covers the largest portion of Africa according to the global map\"; according to the figure, detection in Africa does not look more prevalent compared to other continents. Finally, the text mentions detection in South Africa, Algeria, Rwanda and Uganda, while the maps shows South Africa, Algeria, Tunisia and Egypt. The last section, \"The role of high-throughput sequencing …\" is very generic, and barely addresses how this applied to colistin resistance, as the tile of the review suggests. It contain very general statements about the possibilities of HTS for AMR detection, that have been reviewed in more details in a few recent publications. The authors should take the opportunity of assessing how HTS led to the rapid detection of the spread of the newly identified colistin resistance risk, in a way that was not possible before. Relevant studies should include, among others, the following:\n\nHasman, H., Hammerum, A.M., Hansen, F., Hendriksen, R.S., Olesen, B., Agersø, Y., Zankari, E., Leekitcharoenphon, P., Stegger, M., Kaas, R.S., et al. (2015). Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat1. Kluytmans–van den Bergh, M.F., Huizinga, P., M. J. Bonten, M. Bos, K. De Bruyne, A. W. Friedrich, J. W. Rossen, P. H. Savelkoul, and J. A. Kluytmans. (2009) Presence of mcr-1-positive Enterobacteriaceae in retail chicken meat but not in humans in the Netherlands since 20092. Yu, C. Y., Ang, G. Y., Chong, T. M., Chin, P. S., Ngeow, Y. F., Yin, W. F., & Chan, K. G. (2016). Complete genome sequencing revealed novel genetic contexts of the mcr-1 gene in Escherichia coli strains3.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\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": [ { "c_id": "4646", "date": "20 May 2019", "name": "Dickson Aruhomukama", "role": "Author Response", "response": "We appreciate all the comments made by the reviewer, these helped us to make the manuscript even much better. In the revised manuscript we addressed the comments made as follows; Minor comments – (i) we respect the opinion of the reviewer in regards to reworking the sections, however, we noted that this would grossly distort the manuscript and hence choose to maintain the order as is. We agree with the concerns raised in comments (ii), (iii) and, (iv), we addressed each of these as reflected in version 2 of the manuscript. Major comments - we agree with the reviewer's comments in regards reworking the section on Africa (i.e. comments i, ii and, iii), these have all been addressed in version 2 of the manuscript. In regards to comment (iv), we reviewed the articles suggested by the reviewer and added a section that briefly describes how HTS leads to the rapid detection of the spread of the newly identified colistin resistance risk. The articles were referenced as well." } ] }, { "id": "47772", "date": "29 Apr 2019", "name": "Hosam Mamoon Zowawi", "expertise": [ "Reviewer Expertise Antimicrobial resistance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Mboowa et al focuses on reviewing the molecular mechanisms underlying the role of Colistin resistance in and the importance of high-throughput sequencing and data analysis in identifying chromosomal and plasmid resistance. The authors explored the spread of colistin resistance genes and gave an example on the spread of mcr-1 in Africa. They gave examples of the lipid A modification to be linked to two component systems. Moreover, they demonstrated the link between other resistance pathways and colistin where they highlighted examples of mobile colistin elements and tetracycline and carbapenem resistance co-transfer and co-exist. Finally, they described the role of High-throughput sequencing technologies and identified several platforms and methods. Overall, the review is interesting and the data presented in the manuscript is somewhat supporting the conclusion. However, there are a few comments that need to be addressed prior to indexing:\n\nGeneral comments:\nThe manuscript is written in poor English. Several statements are too long and lack clarity, precision, and completeness. The manuscript has to be rewritten in proper English before submitting it for a detailed revision. As an example “The modifications of the lipid A, which reduce and/or abolish the initial charge-based interaction with the polymyxins in bacteria13,15,16 and also the addition of either/or the 4-amino-4- deoxy-L-arabinose (L-Ara4N) and the phosphoethanolamine (PEtn) that ultimately form the basis of colistin resistance in bacteria16, is mediated by chromosomally encoded genes” *Very confusing and needs further explanations. Authors need to spell out the abbreviation as commonly used. As an example HTS should be NGS (next generation sequencing) in the manuscript. In the introduction section, the authors adapted figure 1 of Africa, which talks about countries are not represented in figure 1 and the figure is not matching what is written. Perhaps a different figure could represent the text better. The reference quoted need to be checked as I have noticed addition of unnecessary references or references that do not match the text. Example reference 4, 16, 17 are quoted in some places where it’s not needed have 3 references and do not match the text. The abstract mentions novel mobile colistin resistance plasmid that was not discussed.\nSpecific comments:\nExamples of bacterial type and specific chromosomal mutations are needed, generalization of the resistance pattern could be misleading especially with chromosomal genes. Also, other known chromosomal genes such as pmrC, crrA/B/C, dedA. Finally, as an example a gene such as eraRis known to be linked to colistin only in Pseudomonasas a response regulator, therefore, the authors are encouraged to mention that different mutations in different organisms can be identified by NGS. Repetition of mcr-1,mcr-2 and mcr-3 sentences several times and not including the rest of the genes all the way to the newly discovered but not published yet mcr-9 and maybe mention the location of such genes.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? No\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "4647", "date": "20 May 2019", "name": "Dickson Aruhomukama", "role": "Author Response", "response": "We appreciate all the comments made by the reviewers, these helped us to make the manuscript even much better. In the revised manuscript we addressed the comments made as follows; General comments – we totally agree with comment (i) in regards to a number of sections being too long and, lacking clarity, precision, and completeness, we have reviewed the entire manuscript, identified sections with these concerns and have endeavored to make these shorter, more clear, precise and, complete. In regards to comment (ii), we agree that HTS and WGS are interchangeably used, however, since we used HTS in the manuscript we have to choose to maintain the use of HTS. Changes were made to address comment (iii), (iv) and, (v) as well. Specific comments – (i) we agree that not all chromosomally mediated resistance mechanisms were discussed, however, we ultimately highlight the potential of WGS in the identification of these as well as acquired resistance mechanisms. Comment (ii) was addressed as well." } ] } ]	1	https://f1000research.com/articles/8-150
https://f1000research.com/articles/5-2674/v1	16 Nov 16	{ "type": "Method Article", "title": "A predictive model of overall survival in patients with metastatic castration-resistant prostate cancer", "authors": [ "Mehrad Mahmoudian", "Fatemeh Seyednasrollah", "Liisa Koivu", "Outi Hirvonen", "Sirkku Jyrkkiö", "Laura L. Elo", "Fatemeh Seyednasrollah", "Liisa Koivu", "Outi Hirvonen", "Sirkku Jyrkkiö", "Laura L. Elo" ], "abstract": "Metastatic castration resistant prostate cancer (mCRPC) is one of the most common cancers with a poor prognosis. To improve prognostic models of mCRPC, the Dialogue for Reverse Engineering Assessments and Methods (DREAM) Consortium organized a crowdsourced competition known as the Prostate Cancer DREAM Challenge. In the competition, data from four phase III clinical trials were utilized. A total of 1600 patients’ clinical information across three of the trials was used to generate prognostic models, whereas one of the datasets (313 patients) was held out for blinded validation. As a performance baseline, a model presented in a recent study (so called Halabi model) was used to assess improvements of the new models. This paper presents the model developed by the team TYTDreamChallenge to predict survival risk scores for mCRPC patients at 12, 18, 24 and 30-months after trial enrollment based on clinical features of each patient, as well as an improvement of the model developed after the challenge. The TYTDreamChallenge model performed similarly as the gold-standard Halabi model, whereas the post-challenge model showed markedly improved performance. Accordingly, a main observation in this challenge was that the definition of the clinical features used plays a major role and replacing our original larger set of features with a small subset for training increased the performance in terms of integrated area under the ROC curve from 0.748 to 0.779.", "keywords": [ "prostate cancer", "mCRPC", "boosting", "survival analysis" ], "content": "Introduction\n\nProstate cancer is the second most common cancer according to the World Cancer Report 20141. Hence it is one of the most studied cancer types with focus on diagnosis and prognosis. A major cause of death among prostate cancer patients is the development of metastatic castrate-resistant prostate cancer (mCRPC), which is both a persistent as well as progressing disease resistant to androgen deprivation therapy2.\n\nIn order to boost research regarding prostate cancer, a crowdsourced competition was designed by the Dialogue for Reverse Engineering Assessments and Methods (DREAM) Consortium in collaboration with Project Data Sphere LLC (PDS) to improve prognostic models of mCRPC. Using data from four phase III clinical trials available through PDS, two main sub-challenges were designed. Sub-challenge 1 was aimed at improving prediction of survival risk for mCRPC patients, whereas Sub-challenge 2 was intended to predict adverse events in patients treated with docetaxel, the standard of care for mCRPC patients at the time of the trials. This paper presents the model developed by the team TYTDreamChallenge in Sub-challenge 1 to predict survival risk scores for mCRPC at 12, 18, 24 and 30-months after diagnosis based on clinical features of each patient, as well as some post-challenge analysis to improve our initial model.\n\nVarious prognostic models for mCRPC have been previously developed3–6. Recently, Halabi et al. developed a prognostic model for mCRPC using eight clinical features (Eastern Cooperative Oncology Group performance status (ECOG), disease site, lactate dehydrogenase, opioid analgesic use, albumin, hemoglobin, prostate-specific antigen, and alkaline phosphatase) and validated it on an external dataset6. The aim of the Prostate Cancer DREAM Challenge was to develop and validate new prognostic models that improve on this current gold-standard Halabi model6.\n\nIn this paper an implementation of generalized boosted models in the form of an R7 package named gbm (generalized boosted regression models) was used to predict overall survival of mCRPC patients using a Cox proportional hazard model as the underlying regression model8. This package is an extension to Freund and Schapire's AdaBoost algorithm9 and Friedman's gradient boosting machine10. In general, boosting is a concept in supervised machine learning with the goal of generating multiple relatively weak learner models, each of which individually works slightly better than random guess, and use them all in corporation to have a highly accurate overall model11.\n\n\nMethods\n\nThe methodology used by our team consisted of two major steps12. The first step was data preparation in which some features were removed from the study due to missing values, high correlations with other features or being unimportant for such survival analysis as determined by clinical experts. The second step was model building utilizing generalized boosted models.\n\nThe data used in this study was collected from mCRPC patients by four institutes. The datasets were based on a cancer treatment trial in which patients received docetaxel treatment. Details of the four trials are shown in Table 1. In the Prostate Cancer DREAM Challenge three (ASCENT-213, MAINSAIL14 and VENICE15) out of the four datasets were available as training sets. The remaining dataset (ENTHUSE-3316) was used for validation by the DREAM Challenge organizers without releasing the survival data to the participants of the competition. All the data were gathered into five major tables (Supplementary Table 1). Additionally, a sixth table, called CoreTable, was provided by the challenge organizers. The CoreTable is a collection of features from the other five tables that summarized the baseline (day 0) values. The clinical features in CoreTable contain treatment variables, cancer staging based on AJCC17, Gleason Score18, ECOG Performance Status9 and lesion details. This table was curated by challenge organizers and was considered as the main table of this challenge.\n\nOut of all the data provided by the Prostate Cancer DREAM Challenge organizers (Supplementary Table 1), we focused on the CoreTable and LabValue tables to form the training and validation datasets. The LabValue table is an event level longitudinal data table which contains all the lab tests performed along with the sampling date and reference range of each lab test. The CoreTable consists of 131 features, of which two are for identification, five are dependent variables and 124 are independent variables. The two dependent variables we used in this study were DEATH and LKADT_P (time to event). The former indicates the death status of a patient and has value “YES” for patients who have died from mCRPC and value “NO” otherwise. The latter is the last day that the patient was known to be alive. Additionally, we processed and extracted further information from the LabValue table to complement the CoreTable.\n\nThe full set of Challenge data is available under the standard Synapse Terms and Conditions of Use and the Prostate Cancer DREAM Challenge Rules and can be downloaded from Synapse web interface. The links and authentication information are available in the following URL:\n\nhttps://www.synapse.org/ProstateCancerChallenge\n\nProcessing of the laboratory values (table LabValue) consisted of a sequence of actions. First, it was observed that there were some duplicate rows in the data; hence 2545 rows were removed. Secondly, based on consultation with oncologists, rows with measurements of 13 important lab tests were extracted, including ALT, AST, ALP, LDH, MG, PHOS, ALB, TPRO, PSA, HB, WBC, NEU and LYM (Table 2). After this step, the number of rows left in the data was 80744. Thirdly, we removed 603 rows marked with “NOT DONE” status in the LBSTAT column, which specifies completion status of the lab test, and with missing value in their LBSTRESC column, which contains standardized format of the test results. Finally, only the 17015 baseline measurements from the 1599 patients were kept in the study, while removing the other follow-up measurements over time as they were unavailable in the validation data. During the steps explained above, one patient (ASC-518-0003) was completely removed from the analysis because of having “NOT DONE” status in all of the important lab tests including ALT, AST, ALP and LDH.\n\nThe measurement values for all lab tests, except PSA, were standardized based on their minimum and maximum ranges as\n\n\n\nwhere x is the observed value of the lab test and β and α are the corresponding upper and lower limit of the reference range. The standardized values are between -1 and 1 if the lab test value is within the normal range. The PSA values were only log2 transformed and the issue of log20 was bypassed by adding e-4 to the values before log2 transformation. The ALP and NEU values were truncated to 10 and 5, respectively. Finally, HB values and log2 transformed AST values were copied from the CoreTable, since these two features contained numerous missing values in the LabValue table.\n\nIn the validation dataset, there were two patients (AZ-00131 and AZ-00383) that had no records in the LabValue table nor in the CoreTable. To predict their survival using the laboratory values, we extracted medians of those 13 features across all patients and used them for these two patients.\n\nIn addition to lab measurements, we considered some additional features from the CoreTable. These included ECOG_C and ANALGESICS as well as four derived features that were summarized to reduce the variation and existing noise in the data. These included LESIONS, DRUGS, DISEASES and PROCEDURES, which were defined as arithmetic sums of the numbers of lesions, medicines, diseases or medical operations, respectively. LKADT_P and DEATH were also directly adopted from CoreTable, which denote the survival time and survival event respectively.\n\nAs the final step in pre-processing, the resulting training and validation datasets were checked for features having large proportions of missing values or having missing values for a particular data provider. The missingness in data is shown in Supplementary Figure 1A. Based on this, seven features including MG, ALB, TPRO, LYM, PHOS, LDH and ALT were excluded from the training and validation sets. Additionally, to minimize the number of highly correlated features in the training data, we further removed the feature WBC, which showed high correlation with NEU (Supplementary Figure 1B).\n\nAt the end of the pre-processing, the training set consisted of 1599 patients and validation set of 313 patients. Both datasets had 15 features out of which two were for identification, two were response features and the other 11 were independent predictor variables.\n\nTo develop a model of overall survival in mCRPC, we utilized a gradient boosting algorithm based on regression trees, with a Cox proportional hazard model as the underlying regression model. The R package gbm20 was used with 5000 trees, 10 fold cross-validation, minimum 3 observations in the trees’ terminal nodes and step-size reduction value of 0.007.\n\nIn the DREAM Challenge competition, we submitted a separate risk score for each patient in 12, 18, 24 and 30 months. For 18, 24 and 30 months, modeling was done individually for each data provider, and the mean of the three individual risk score predictions was then calculated as the final risk score at each time point. For 12 months survival, all the training data were used to create a single model and a risk score prediction. After the challenge, we also tested the performance of the models when determining only a single overall risk score for each patient. For this purpose, two strategies were considered for the post-challenge analysis: 1) average of risk scores obtained separately for each data provider (referred to as PostSeparate), or 2) a single risk score obtained by combining data from all the providers in the modelling (referred to as PostCombined).\n\nThe performance of the predictions was measured using the integrated area under the ROC curve (iAUC) from 6 to 30 months, as well as separate AUC values at 12, 18, and 24 months. The iAUC was calculated using the R package timeROC (version 0.3)21. The performance measures were obtained from blinded validation by the DREAM organizers.\n\n\nResults and discussion\n\nThe performance of the TYTDreamChallenge model (iAUC=0.748) was significantly better than random. However, it did not perform statistically significantly better than the gold-standard Halabi model (iAUC=0.743, Bayes factor < 3), as determined by the DREAM organizers22.\n\nTo further investigate the possibility to improve our model after the challenge, we considered in our post-challenge analysis the impact of calculating an overall risk score instead of our original strategy of having separate scores for the different time points. Interestingly, this had a marked effect on the performance of our model (Figure 1). When the average model across the different data providers was considered, the iAUC improved to 0.757 (model PostSeparate). When all the data were used together for model building, the iAUC increased further to 0.777 (model PostCombined).\n\nThe integrated area under the ROC curve (iAUC) from 6 to 30 months, as well as separate AUC values at 12, 18, and 24 months are shown. The performance measures were obtained from blinded validation by the DREAM organizers. The first and second team refer to the top-ranked models in the DREAM Challenge; PostCombined and PostSeparate refer to two different post-challenge analyses using the same modelling strategy and same features as in our original DREAM Challenge submission (TYTDreamChallenge) but, instead of having time-specific models, a single overall risk score was calculated for each patient either as an average risk score across the data providers (PostSeparate) or as a single risk score obtained by combining data from all the providers in the modelling (PostCombined).\n\nNext, we examined the relative importance of the different features on the predictions in the PostCombined model, as determined by the boosting algorithm (Figure 2A). As expected, many of the features used in the Halabi model had high importance also in our model (PSA, ALP, HB). However, additional features were found (AST, NEU). On the other hand, ECOG_C was not as important in our model as it was in the Halabi model. We also tested the effect of removing one variable at a time when building the model (Figure 2B). This supported further the importance of ALP, HB, AST, PSA and LESIONS, whereas the removal of NEU actually improved the performance further (iAUC=0.780). Removal of PROCEDURES, ANALGESICS, ECOG_C, DISEASES or DRUGS did not have a marked impact on the performance.\n\n(A) Relative influence in the post-challenge model where a single overall risk score was calculated for each patient by combining data from all the providers in the modelling (PostCombined). (B) Effect of removing one feature at a time when building the model. The integrated area under the ROC curve (iAUC) from 6 to 30 months, as well as separate AUC values at 12, 18, and 24 months are shown. The performance measures were obtained from blinded validation by the DREAM organizers.\n\nFinally, we applied the same boosting strategy to build a model using only five features ALP, HB, LESIONS, AST and PSA (Figure 3A; referred to as PostFive). Notably, the performance in the validation data did not decrease markedly from that with a larger set of features (iAUC=0.779). Among the features, PSA and ALP had the largest relative importance in predicting the survival, whereas LESIONS had the lowest relative importance (Figure 3B). To assist in understanding the contribution of the identified features, partial dependence plots were examined, which illustrate the partial dependence of the risk scores on each feature after accounting for the effects of the other features. This suggested intuitive interpretations for the different features (Figure 3C). Similarly as in the Halabi model, the risk increases with high values of PSA and ALP, high numbers of LESIONS, and low values of HB6. Additionally, our model suggests that high values of AST increase the risk. These findings are well in line with the general hypothesis that these factors are basic values representing the volume of the disease.\n\n(A) Performance of the boosting strategy using only five features ALP, HB, LESIONS, AST and PSA, as compared to the DREAM Challenge models and our post-challenge models. The integrated area under the ROC curve (iAUC) from 6 to 30 months, as well as separate AUC values at 12, 18, and 24 months are shown. The performance measures were obtained from blinded validation by the DREAM organizers. (B) Relative importance of the different features on the predictions. (C) Partial dependence plots illustrating the partial dependence of the risk scores on each feature after accounting for the effects of the other features.\n\nTaken together, based on the blindly validated submissions it can be concluded that the proposed post-challenge model in this paper (PostCombined) is markedly better than the gold-standard Halabi model. The post-challenge analysis revealed that a single overall risk score performs better than our original strategy of time-specific risk scores by better targeting the overall survival patterns of patients. A model based on only five features ALP, HB, AST, PSA and LESIONS produced a relatively high accuracy compared to the Halabi model with eight features or the model of the winning team involving a large number of features and their interactions. Thus the five feature model presented here provides an efficient option in terms of practical clinical use.\n\nThe present study focused on clinical features only. Additional possibilities to improve the performance of the models would be to add molecular level information, such as gene expression data, to training and test sets.\n\n\nData and software availability\n\nThe Challenge datasets can be accessed at: https://www.projectdatasphere.org/projectdatasphere/html/pcdc\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/ProstateCancerChallenge\n\nThe code and documentation underlying the method presented in this paper can be found at: http://dx.doi.org/10.5281/zenodo.4770623\n\nThe latest source code is available at: https://bitbucket.org/mehrad_mahmoudian/dream-prostate-cancer-challenge-q.1a", "appendix": "Author contributions\n\n\n\nMM participated in the pre-processing of the data, performed all the post-challenge analyses and drafted the manuscript. FS pre-processed the data and developed the TYTDreamChallenge model. LK, OH and SJ participated in the pre-processing and provided the clinical insights. LLE designed and supervised the study, participated in the analyses and drafted the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by the Sigrid Juselius Foundation (to L.L.E) and the University of Turku Graduate School (to F.S).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThis publication is based on research using information obtained from www.projectdatasphere.org, which is maintained by Project Data Sphere, LLC. Neither Project Data Sphere, LLC nor the owner(s) of any information from the web site have contributed to, approved or are in any way responsible for the contents of this publication.\n\nWe would also thank the Sage Bionetworks, the DREAM organization, and Project Data Sphere for developing and supplying data for the Challenge.\n\n\nSupplementary material\n\n(A) Proportions of missing values for the 13 lab tests selected on the basis of consultation with oncologists. The proportion of missing values is shown separately for each clinical trial dataset (columns), with red indicating large proportions of missing values. (B) Pearson correlation between the different features across all studies. The darker the color, the higher the correlation.\n\n\nReferences\n\nStewart B, Wild CP (eds.): World Cancer Report 2014. I. A. for R. on C. W. 2014. Reference Source\n\nHotte SJ, Saad F: Current management of castrate-resistant prostate cancer. Curr Oncol. 2010; 17(Suppl 2): S72–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLucarelli G, Ditonno P, Bettocchi C, et al.: Serum sarcosine is a risk factor for progression and survival in patients with metastatic castration-resistant prostate cancer. Future Oncol. 2013; 9(6): 899–907. PubMed Abstract \| Publisher Full Text\n\nPond GR, Armstrong AJ, Galsky MD, et al.: Efficacy of docetaxel-based chemotherapy following ketoconazole in metastatic castration-resistant prostate cancer: implications for prior therapy in clinical trials. Urol Oncol. 2013; 31(8): 1457–63. PubMed Abstract \| Publisher Full Text\n\nQu YY, Dai B, Kong YY, et al.: Prognostic factors in Chinese patients with metastatic castration-resistant prostate cancer treated with docetaxel-based chemotherapy. Asian J Androl. 2013; 15(1): 110–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalabi S, Lin CY, Kelly WK, et al.: Updated prognostic model for predicting overall survival in first-line chemotherapy for patients with metastatic castration-resistant prostate cancer. J Clin Oncol. 2014; 32(7): 671–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. 2015. Reference Source\n\nCox DR: Regression Models and Life-Tables. J R Stat Soc Ser B. 1972; 34(2): 187–220. Reference Source\n\nFreund Y, Schapire R, Abe N: A short introduction to boosting. Journal-Japanese Soc Artif Intell. 1999; 14(5): 1612. Reference Source\n\nFriedman JH: Greedy Function Approximation: A Gradient Boosting Machine. Ann Stat. 2001; 29(5): 1189–1232. Publisher Full Text\n\nSchapire RE: The strength of weak learnability. Mach Learn. 1990; 5(2): 197–227. Publisher Full Text\n\nTYT Prostate Cancer DREAM Challenge writeup - syn4228911. Reference Source\n\nScher HI, Jia X, Chi K, et al.: Randomized, open-label phase III trial of docetaxel plus high-dose calcitriol versus docetaxel plus prednisone for patients with castration-resistant prostate cancer. J Clin Oncol. 2011; 29(16): 2191–2198. PubMed Abstract \| Publisher Full Text\n\nPetrylak DP, Vogelzang NJ, Budnik N, et al.: Docetaxel and prednisone with or without lenalidomide in chemotherapy-naive patients with metastatic castration-resistant prostate cancer (MAINSAIL): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Oncol. 2015; 16(4): 417–425. PubMed Abstract \| Publisher Full Text\n\nTannock IF, Fizazi K, Ivanov S, et al.: Aflibercept versus placebo in combination with docetaxel and prednisone for treatment of men with metastatic castration-resistant prostate cancer (VENICE): a phase 3, double-blind randomised trial. Lancet Oncol. 2013; 14(8): 760–8. PubMed Abstract \| Publisher Full Text\n\nFizazi K, Higano CS, Nelson JB, et al.: Phase III, randomized, placebo-controlled study of docetaxel in combination with zibotentan in patients with metastatic castration-resistant prostate cancer. J Clin Oncol. 2013; 31(14): 1740–1747. PubMed Abstract \| Publisher Full Text\n\nSchmoll HJ, Greene FL, Page DL, et al. (eds): AJCC Cancer Staging Manual, 6th edition. Ann Oncol. 2003; 14(2): 345–346. Publisher Full Text\n\nGleason DF, Mellinger GT: Prediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging. J Urol. 1974; 111(1): 58–64. PubMed Abstract \| Publisher Full Text\n\nOken MM, Creech RH, Tormey DC, et al.: Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982; 5(6): 649–55. PubMed Abstract \| Publisher Full Text\n\nRidgeway G: gbm: Generalized Boosted Regression Models. 2015. Reference Source\n\nBlanche P, Dartigues JF, Jacqmin-Gadda H: Estimating and Comparing time-dependent areas under receiver operating characteristic curves for censored event times with competing risks. Stat Med. 2013; 32(30): 5381–5397. PubMed Abstract \| Publisher Full Text\n\nDREAM9.5 - Prostate Cancer DREAM Challenge - syn2813558. 2015. Reference Source\n\nMahmoudian M, Seyednasrollah F, Koivu L, et al.: Source code for “A predictive model of overall survival in patients with metastatic castration-resistant prostate cancer”. 2016. Data Source" }	[ { "id": "23226", "date": "26 Jun 2017", "name": "Motoki Shiga", "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 paper offers a survival time prediction method based on a gradient boosting algorithm for a Cox proportional hazard model. The analysis of the Prostate Cancer DREAM Challenge dataset found that the prediction performance of a model built from all three clinical trials is much better than the performance of models built from each clinical trial.\n\nMajor comments:\n\nAdditional comparison with the prediction performance of the Halabi model built from all clinical trials and the performance of the models built using each trial should help to evaluate method PostCombined in more detail. And the description of procedures of the first and second winning teams is cessary.\nWhat is the test data to compute iAUC? It was clinical trial AstraZeneca in the challenge. The manuscript should describe the procedure of the performance evaluation in detail.\n\nMinor comments:\n\nPlease explain the machine learning method used in this work in more detail.\n\nThe threshold value of the feature selection should be described in the third paragraph of the right column in page 4.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "23094", "date": "26 Jun 2017", "name": "Peter K Rogan", "expertise": [ "Reviewer Expertise Machine learning", "mathematical modeling", "gene signature analysis", "cancer 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\nIn general, the paper is understandable and well organized. The use of boosting to improve the Cox proportional hazard and regression models is interesting. Despite the issues the authors address regarding missing data, there appears to be adequate information in this dataset to test this approach.\n\nAbstract\n“as well as an improvement of the model developed after the challenge” This is vague statement that adds little to the abstract. It is irrelevant when the improvement was made. The abstract should describe the clinical features and any improvements in an organized manner so that the reader can determine whether the method proposed differs from previous approaches.\n\n“TYTDreamChallenge model performed similarly as the gold-standard Halabi model” What was the performance accuracy or misclassification rate?\nThe “Halabi” model terminology is jargonistic and should be described briefly in conventional oncology criteria (ie. an abbreviated form of the statement in the introduction would be sufficient). Was the increase in the ROC by 3% significant? If so, please explain why.\nIntroduction\nThe authors term the paper by Halabi et al. to be the “gold standard” prognostic model. While it performs well and is a reasonable comparator, it does not meet the criteria for a gold standard. It is a conventionally trained and tested and validated with a single external dataset. Gold standards, on the other hand, have been reproduced by other investigators using other patient cohorts multiple times with similar findings, may have fulfilled ISO or other standards and have been recommended by internationally recognized authorities (for example, the dicentric chromosome assay for radiation dosimetry).\n\nMethods\n“some features were removed from the study due to missing values, high correlations with other features or being unimportant for such survival analysis as determined by clinical experts. “ Please indicate which features were removed. What proportions were attributable to missing values, etc.\n“based on consultation with oncologists, rows with measurements of 13 important lab tests” Define important vs unimportant lab tests, and reasons for selecting them.\n\nDid the authors evaluate whether their methods were very sensitive to assumptions made about the missingness mechanism or about the distributions of the variables with missing data? If so, please state.\nResults\nThe authors don’t clearly distinguish which methods were used in their submission of results to the DREAM challenge vs. how or why the “improvements” were made after the submission to the challenge. Furthermore, the overall risk score vs separate scores at different time points can simply be reported, without making this artificial distinction. Thus, the distinction made in the abstract between these lacks context, and I would recommend removing it.\n\nThere are many other measures for evaluating the models that the authors could report besides AUC, including Matthews Correlation Coefficient, F-measure, Precision, and Accuracy. They may consider computing and reporting these.\n\nFigure 3 is unacceptable quality. Even at 200% magnification, the labels on each of the graphs are barely readable. Panel C requires some further explanation in order to interpret it. The text indicates “This suggested intuitive interpretations for the different features,” which does not explain the results or whether the partial dependence can be used for feature selection or interpretation.\n\n“These findings are well in line with the general hypothesis that these factors are basic values representing the volume of the disease.” By volume, are the authors referring to the extent of the disease? The extent of the disease is not the same as the survival risk, which is what the authors state they are modeling in the introduction.\n\nIn cancer, volume refers to the size of the tumor and quantitative distribution (Castro-Mesta et al. 20161). In fact, the authors use the numbers of lesions to infer survival risk, so it would appear that this is a circular argument.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "4594", "date": "17 May 2019", "name": "Mehrad Mahmoudian", "role": "Author Response", "response": "We would like to thank the reviewer for the time and effort he has put into our manuscript and the insightful comments. We have addressed all the comments in this response as well as in the main text and figures accordingly. Abstract > “as well as an improvement of the model developed after the challenge” > This is vague statement that adds little to the abstract. It is irrelevant when the improvement was made. The abstract should describe the clinical features and any improvements in an organized manner so that the reader can determine whether the method proposed differs from previous approaches. Thank you for your comment. We understand your concern that the text was not clear enough to convey the concept we wanted to the full extent. Therefore, we have modified the text to improve clarity and transparency and included information about the clinical features in the abstract as suggested by the reviewer. Regarding the time of analysis, we feel that it should be mentioned to comply with the DREAM Challenge procedure and evolution of developing the ultimate model. > “TYTDreamChallenge model performed similarly as the gold-standard Halabi model” > What was the performance accuracy or misclassification rate? All the validations of the predictions were done through submitting the predicted values to the DREAM Challenge evaluation system as have been explained in detail in https://www.synapse.org/#!Synapse:syn2813558/wiki/209591 . As the true response values of the validation dataset were kept hidden from the participants, unfortunately, the requested information cannot be provided. We have modified the text to clarify this fact accordingly. > The “Halabi” model terminology is jargonistic and should be described briefly in conventional oncology criteria (ie. an abbreviated form of the statement in the introduction would be sufficient). Was the increase in the ROC by 3% significant? If so, please explain why. We have clarified the term “Halabi model” in the abstract via: “The previously introduced prognostic model of overall survival of chemotherapy-naive mCRPC patients treated with docetaxel or prednisone (so called Halabi model) was used as a performance baseline.” Regarding the comment about significance, unfortunately as explained above, we do not have access to the true response values and therefore the significance between the ROC curves cannot be computed. Introduction > The authors term the paper by Halabi et al. to be the “gold standard” prognostic model. While it performs well and is a reasonable comparator, it does not meet the criteria for a gold standard. It is a conventionally trained and tested and validated with a single external dataset. Gold standards, on the other hand, have been reproduced by other investigators using other patient cohorts multiple times with similar findings, may have fulfilled ISO or other standards and have been recommended by internationally recognized authorities (for example, the dicentric chromosome assay for radiation dosimetry). We agree that the term “gold-standard” was not a good choice. Therefore, we have changed all instances to “performance baseline”. Methods > “some features were removed from the study due to missing values, high correlations with other features or being unimportant for such survival analysis as determined by clinical experts. “ > Please indicate which features were removed. What proportions were attributable to missing values, etc. We agree that the description of the feature selection process was not very clear. We have now modified the text. The revised Supplementary Figure 1 now shows the proportions of missing values and the correlations between the 13 lab tests considered. We also provide the source codes of this study publicly available, containing the in-depth details of the feature exclusion along with the exact procedure to reproduce the results. The DOI of the published code is doi:10.5281/zenodo.47706 which is also presented as the last reference of the manuscript. > “based on consultation with oncologists, rows with measurements of 13 important lab tests” > Define important vs unimportant lab tests, and reasons for selecting them. “Important” referred to the educated estimation of the oncologists about the importance of investigating these features. We have now rephrased the text. > Did the authors evaluate whether their methods were very sensitive to assumptions made about the missingness mechanism The features used in the modelling contained only relatively few missing values. Therefore, in the current study, we did not investigate the missingness mechanisms. Results > The authors don’t clearly distinguish which methods were used in their submission of results to the DREAM challenge vs. how or why the “improvements” were made after the submission to the challenge. Furthermore, the overall risk score vs separate scores at different time points can simply be reported, without making this artificial distinction. Thus, the distinction made in the abstract between these lacks context, and I would recommend removing it. The main difference between the original submission and the post-challenge submission was a more systematic feature selection approach in the latter. This is now clarified in the revised manuscript. According to the suggestion by the Reviewer, we have now removed from the abstract the distinction between the overall risk score vs separate scores at different time points. > There are many other measures for evaluating the models that the authors could report besides AUC, including Matthews Correlation Coefficient, F-measure, Precision, and Accuracy. They may consider computing and reporting these. The true response values of the independent validation set are not publicly available. Therefore, it is not possible to calculate other performance measures than those available from the DREAM evaluation system. > Figure 3 is unacceptable quality. Even at 200% magnification, the labels on each of the graphs are barely readable. We agree that the font size could be larger and therefore we have increased the font size in Figure 3. > Panel C requires some further explanation in order to interpret it. The text indicates “This suggested intuitive interpretations for the different features,” which does not explain the results or whether the partial dependence can be used for feature selection or interpretation. We agree that the legend did not address the interpretation of the partial dependence plots. Therefore, we have completely revised the text for panel C and have elaborated on the interpretation. > “These findings are well in line with the general hypothesis that these factors are basic values representing the volume of the disease.” > By volume, are the authors referring to the extent of the disease? The extent of the disease is not the same as the survival risk, which is what the authors state they are modeling in the introduction. In cancer, volume refers to the size of the tumor and quantitative distribution (Castro-Mesta et al. 20161). In fact, the authors use the numbers of lesions to infer survival risk, so it would appear that this is a circular argument. We agree with the reviewer that the term “volume” was not correct in this sentence. We have modified the sentence accordingly." } ] } ]	1	https://f1000research.com/articles/5-2674
https://f1000research.com/articles/8-152/v1	05 Feb 19	{ "type": "Software Tool Article", "title": "TFutils: Data structures for transcription factor bioinformatics", "authors": [ "Benjamin J. Stubbs", "Shweta Gopaulakrishnan", "Kimberly Glass", "Nathalie Pochet", "Celine Everaert", "Benjamin Raby", "Vincent Carey", "Benjamin J. Stubbs", "Shweta Gopaulakrishnan", "Kimberly Glass", "Nathalie Pochet", "Celine Everaert", "Benjamin Raby" ], "abstract": "DNA transcription is intrinsically complex. Bioinformatic work with transcription factors (TFs) is complicated by a multiplicity of data resources and annotations. The Bioconductor package TFutils includes data structures and functions to enhance the precision and utility of integrative analyses that have components involving TFs. TFutils provides catalogs of human TFs from three reference sources (CISBP, HOCOMOCO, and GO), a catalog of TF targets derived from MSigDb, and multiple approaches to enumerating TF binding sites. Aspects of integration of TF binding patterns and genome-wide association study results are explored in examples.", "keywords": [ "Transcription factors", "Gene expression", "Gene regulation", "Bioconductor" ], "content": "Introduction\n\nA central concern of genome biology is improving understanding of gene transcription. In simple terms, transcription factors (TFs) are proteins that bind to DNA, typically near gene promoter regions. The role of TFs in gene expression variation is of great interest. Progress in deciphering genetic and epigenetic processes that affect TF abundance and function will be essential in clarifying and interpreting gene expression variation patterns and their effects on phenotype. Difficulties of identifying functional binding of TFs, and opportunities for using information of TF binding in systems biology contexts, are reviewed in Lambert et al.1 and Weirauch et al.2.\n\nThis paper describes an R/Bioconductor package called TFutils, which assembles various resources intended to clarify and unify approaches to working with TF concepts in bioinformatic analysis. Computations described in this paper can be carried out with Bioconductor version 3.8. The package can be installed with\n\n\n\nIn the next section we describe the basic concepts of enumerating and classifying TFs, enumerating TF targets, and representing genome-wide quantification of TF binding affinity. This is followed by a review of the key data structures and functions provided in the package, and an example in cancer informatics.\n\nThe present paper does not deal directly with the manipulation or interpretation of sequence motifs. An excellent Bioconductor package that synthesizes many approaches to these tasks is universalmotif.\n\n\nBasic concepts of transcription factor bioinformatics\n\nGiven the importance of the topic, it is not surprising that a number of bioinformatic research groups have published catalogs of transcription factors along with metadata about their features. Standard nomenclature for TFs has yet to be established. Gene symbols, motif sequences, and position-weight matrix catalog entries have all been used as TF identifiers.\n\nIn TFutils we have gathered information from four widely used resources, focusing specifically on human TFs: Gene Ontology (GO, Ashburner et al.3, in which GO:0003700 is the tag for the molecular function concept “DNA binding transcription factor activity”), CISBP (Catalog of Inferred Sequence Binding Preferences) (Weirauch et al.2), HOCOMOCO (Homo sapiens Comprehensive Model Collection) (Kulakovskiy et al.4), and the “c3 TFT (transcription factor target)” signature set of MSigDb (Molecular Signatures Database) (Subramanian et al.5). Figure 1 depicts the sizes of these catalogs, measured using counts of unique HGNC gene symbols. The enumeration for GO uses Bioconductor’s org.Hs.eg.db (version 3.7.0) package to find direct associations from GO:0003700 to HGNC symbols. The enumeration for MSigDb is heuristic and involves parsing the gene set identifiers used in MSigDb for exact or close matches to HGNC symbols. For CISBP and HOCOMOCO, the associated web servers provide easily parsed tabular catalogs.\n\nAs noted by Weirauch et al.2, interpretation of the “function and evolution of DNA sequences” is dependent on the analysis of sequence-specific DNA binding domains. These domains are dynamic and cell-type specific (Gertz et al.6). Classifying TFs according to features of the binding domain is an ongoing process of increasing intricacy. Figure 2 shows excerpts of hierarchies of terms related to TF type derived from GO (on the left) and TFclass (Wingender et al.7). There is a disagreement between our enumeration of TFs based on GO in Figure 1 and the 1919 shown in AmiGO, as the latter includes a broader collection of receptor activities.\n\nTable 1 provides examples of frequently encountered TF classifications in the CISBP and HOCOMOCO catalogs. The numerical components of the HOCOMOCO classes correspond to TFClass subfamilies (Wingender et al.7).\n\nEntries in columns Nc (Nh) are numbers of distinct TFs annotated to classes in columns CISBP (HO-COMOCO) respectively. Entries are ordered top to bottom by frequency of occurrence. There is no substantive correspondence between entries on a given row. Harmonization of class terminology is beyond the scope of this paper.\n\nThe Broad Institute MSigDb (Subramanian et al.5) includes a gene set collection devoted to cataloging TF targets. We have used Bioconductor’s GSEABase package (version 1.45.0) to import and serialize the gmt representation of this collection.\n\n\n\nNames of TFs for which target sets are assembled are encoded in a systematic way, with underscores separating substrings describing motifs, genes, and versions. Some peculiarity in nomenclature in the MSigDb labels can be observed:\n\n\n\nManual curation will be needed to improve the precision with which MSigDb TF target sets can be associated with specific TFs or motifs.\n\nIn this subsection we address representation of putative binding sites. First we illustrate how to represent sequence-based affinity measures and the binding site locations implied by these. We then discuss use of results of ChIP-seq experiments for cell-type-specific binding site enumeration.\n\nAffinity scores based on reference sequence. The FIMO algorithm of the MEME suite (Grant et al.8) was used to score the human reference genome for TF binding affinity for 689 motif matrices to which genes are associated. Full details are provided in Sonawane et al.9. Sixteen (16) tabix-indexed BED files are lodged in an AWS S3 bucket for illustration purposes.\n\n\n\n\n\nWe harvest scores in a genomic interval of interest (bound to fimo16 in the rowRanges assignment below) using reduceByFile. This yields a list with one element per file. Each such element holds a list of scanTabix results, one per query range.\n\n\n\nscanTabix produces a list of vectors of text strings, which we parse with data.table::fread. The resulting tables are then reduced to a genomic location and -log10 of the p-value derived from the binding affinity statistic of FIMO in the vicinity of that location.\n\n\n\nIt turns out there are too many distinct TFs to display names individually, so we label the scores with the names of the associated TF families as defined in CISBP.\n\n\n\nA simple display of predicted TF binding affinity near the gene ORMDL3 is provided in Figure 3.\n\nPoints are -log10-transformed FIMO-based p-values colored according to TF class as annotated in CISBP. Segments at bottom of plot are transcribed regions of ORMDL3 according to UCSC gene models in build hg19.\n\nTF binding predictions based on ChIP-seq data from ENCODE. The ENCODE project provides BED-formatted reports on ChIP-seq experiments for many combinations of cell type and DNA-binding factors. TFutils includes a table encode690 that gives information on 690 experiments involving pairs formed from 91 cell lines and 161 TFs for which results have been recorded as GRanges instances that can be acquired with the Annotation-Hub (version 2.15.4) package. Positional relationships between cell-type specific binding sites and genomic features can be investigated. An illustration is given in Figure 4, in which is it suggested that in HepG2 cells, CEBPB exhibits a distinctive pattern of binding in the vicinity of ORMDL3.\n\nColored rectangles at top are regions identified as narrow binding peaks, arrows in bottom half are exons in ORMDL3. Arrows sharing a common vertical position are members of the same transcript as cataloged in Ensembl version 75.\n\nWe have compared enumerations of human transcription factors by different projects, provided access to two forms of binding domain classification, and illustrated the use of cloud-resident genome-wide binding predictions. In the next section we review selected details of data structures and methods of the TFutils package.\n\n\nMethods\n\nThe TFutils package is designed to lower barriers to usage of key findings of TF biology in human genome research. TFutils is supplied as a conventional R package distributed with, and making use of, the Bioconductor software ecosystem. TFutils includes ready-to-use reference data, tools for visualizing binding sites, and tools that simplify integrative use of TF binding information with GWAS findings.\n\nCatalogs. Two reference resources have been collected into the TFutils package as data.frame instances. These are cisbpTFcat (CISBP: 7592 x 28), and hocomoco.mono.sep2018 (mononucleotide models, full catalog, 769 x 9). These data.frames are snapshots of the CISBP and HOCOMOCO catalogs\n\nIndexed BED in AWS S3. As described above fimo16 provides programmatic access to FIMO scores for 16 TFs, using the GenomicFiles (version 1.19.0) protocol.\n\nAnnotated reference to ENCODE ChIP-seq results. encode690 simplifies programmatic access to TF:cell-line combinations available in Bioconductor AnnotationHub (version 2.15.4).\n\nTF targets enumerated in MsigDb. The c3-TFT (TF targets) subset from MSigDb is provided as a GeneSet-Collection instance as defined in GSEABase.\n\nIllustrative GWAS records. The full EBI/EMBL GWAS catalog is available in the gwascat package (version 2.15.0); for convenience, an excerpt focusing on chromosome 17 is supplied with TFutils as gwascat_hg19_chr17.\n\nInteractive enumeration of TF targets implicated in GWAS. The TFtargs function runs a shiny app that permits selection of a TF in the nomenclature of the MSigDb c3/TFT gene set collection. The app will search an object provided by the gwascat package for references in the MAPPED_GENE field that match the targets of the selected TF. Figure 5 gives an illustration.\n\nThis example reports on recent EBI GWAS catalog hits on chromosome 17 only.\n\nThe TFCatalog S4 class. Reference catalogs for TF biology are structured with the TFCatalog S4 class. Two essential components for managing a catalog are the native TF identifier for the catalog and the HGNC gene symbol typically used to name the TF. The TFCatalog class includes a name field to name the catalog, and a character vector with elements comprised of the native identifiers for catalogued TFs.\n\nFor example, CISBP uses T004843_1.02 to refer to motifs associated with gene TFAP2B. There are five such motifs, three derived from SELEX, one from Transfac, and one from Hocomoco.\n\nA data.frame instance that has an obligatory column named ‘HGNC’ can include any collection of fields that offer metadata about the TF in the specified catalog. Here is how we construct and view a TFCatalog object using the CISBP reference data.\n\n\n\nThe TFutils package can be installed in any version of R subsequent to 3.5.0, and therefore will be usable on Unix, Windows, or Mac platforms. The preferred method of installation employs the CRAN package BiocManager, through the R command BiocManager::install(\"TFutils\"). All necessary dependencies will be installed through this process.\n\nIn this section we consider applications of the tools in genetic epidemiology. First we look for TFs that may harbor variants associated with traits in the EBI GWAS catalog. Then we show how to enumerate traits associated with targets of a selected TF.\n\nTFs that are direct GWAS hits for a given trait. directHitsInCISBP accepts a string naming a trait, and returns a data.frame of TFs identified as “mapped genes” for the trait, with their TF “family name”.\n\n\n\ntopTraitsOfTargets will acquire the targets of a selected TF, check for hits in these genes in a given GWAS catalog instance, and tabulate the most commonly reported traits.\n\n\n\n\nDiscussion\n\nSources and consequences of variations in DNA transcription are fundamental problems for cell biology, and the projects we have made use of for cataloging transcription factors are at the boundaries of current knowledge.\n\nIt is noteworthy that the four resources used for Figure 1 agree on names of only 119 TFs. The fact that CISBP distinguishes 475 TFs that are not identified in any other source should be better understood. We observe that the ascription of TF status to AHRR is based on its sharing motifs with AHR (see http://cisbp.ccbr. utoronto.ca/TFreport.php?searchTF=T014165_1.02).\n\nFigure 2 and Table 1 show that the classification of TFs is now fairly elaborate. Use of the precise terminology of the TFClass system to label TFs of interest at present relies on associations provided with the HOCOMOCO catalog.\n\nAs population studies in genomic and genetic epidemiology grow in size and scope, principles for organizing and prioritizing loci associated with phenotypes of interest are urgently needed. Figure 5 shows that loci associated with phenotypes related to kidney function, lung function, and IL-8 levels are potentially unified through the fact that the GWAS hits are connected with genes identified as targets of VDR (vitamin D receptor). This example limited attention to hits on chromosome 17; the TFtargs tool permits ad libitum exploration of phenotype-locus-gene-TF associations. Our hope is that the tools and resources collected in TFutils will foster systematic development of evidence-based mechanistic network models for transcription regulation in human disease contexts, thereby contributing to the development of personalized genomic medicine.\n\n\nData availability\n\nWith the exception of the FIMO scoring data (fimo16), all data underlying the results are available as part of the article and no additional source data are required.\n\nfimo16 links to indexed bed files in a public S3 bucket funded by the Bioconductor foundation. The underling data is sourced from Sonawane et al. 2017 https://doi.org/10.1016/j.celrep.2017.10.0019\n\n\nSoftware availability\n\nSource code is available from GitHub: https://github.com/vjcitn/TFutils\n\nArchived source code: https://doi.org/doi:10.18129/B9.bioc.TFutils10\n\nLicence: Artistic License 2.0", "appendix": "Grant information\n\nSupport for the development of this software was provided by the National Institues of Health [U01 CA214846 to VC, U24 CA180996] and the Chan Zuckerberg Initiative [DAF 2018-183436 to VC, R01 NHLBI HL118455 to BR].\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\nLambert SA, Jolma A, Campitelli LF, et al.: The Human Transcription Factors. Cell. 2018; 172(4): 650–665. PubMed Abstract \| Publisher Full Text\n\nWeirauch MT, Yang A, Albu M, et al.: Determination and inference of eukaryotic transcription factor sequence specificity. Cell. 2014; 158(6): 1431–1443. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshburner M, Ball CA, Blake JA, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25(1): 25–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulakovskiy IV, Vorontsov IE, Yevshin IS, et al.: HOCOMOCO: towards a complete collection of transcription factor binding models for human and mouse via large-scale ChIP-Seq analysis. Nucleic Acids Res. 2018; 46(D1): D252–D259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubramanian A, Tamayo P, Mootha VK, et al.: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43): 15545–15550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGertz J, Savic D, Varley KE, et al.: Distinct properties of cell-type-specific and shared transcription factor binding sites. Mol Cell. 2013; 52(1): 25–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWingender E, Schoeps T, Haubrock M, et al.: TFClass: expanding the classification of human transcription factors to their mammalian orthologs. Nucleic Acids Res. 2018; 46(D1): D343–D347. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrant CE, Bailey TL, Noble WS: FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011; 27(7): 1017–1018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSonawane AR, Platig J, Fagny M, et al.: Understanding Tissue-Specific Gene Regulation. Cell Rep. 2017; 21(4): 1077–1088. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarey V, Gopaulakrishnan S: TFutils: TFutils. R package version 1.2.0. 2018. http://www.doi.org/10.18129/B9.bioc.TFutils" }	[ { "id": "44033", "date": "20 Feb 2019", "name": "Lihua Julie Zhu", "expertise": [ "Reviewer Expertise Bioinformatics", "ChIP-seq", "CRISPR technology", "RNA-seq", "annotation", "ATAC-seq", "motif analysis", "shRNA/CRISPR screening", "visualization", "machine learning and database application" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 TFutils package provides useful, convenient, integrated data structures for TF-related bioinformatics analyses, by incorporating the basic information of human transcription factors (TFs), such as TF classification, known TF targets, genome-wide TF binding sites and binding affinity scores, which might be used to prioritize candidate genetic variants and help understand gene transcriptional regulatory mechanisms. Importantly, it also provides an interactive interface to query TFs and TF targets implicated in human traits as discovered by many GWASs. In a quick test, all demo code in this paper worked. However, to make sure TFutils is more useful to the bioinformatics community, a few questions may need to be addressed. Here are our detailed comments and questions.\n\nTFutils includes resources from CISBP, HOCOMOCO, GO and MSigDb. There are additional human TF resources. Is there any reason not to include those resources such as JASPAR1, Transfac2, HDPI3 and uniPro4? There are potential packages that will likely import TFutils such as TFBSTools for the analysis of transcription factor binding sites manipulation, motifStack for graphic representation of multiple motifs5 and MotIV. It will be helpful to present a few lines of code to show how to integrate data from TFutils to aforementioned pipelines. The section “Basic concepts of transcription factor bioinformatics” includes lots of background information, such as existing TF-related data sources/bases, TF classification, and how TFutils incorporates and access those resources. To make it easy to follow, we suggest break this part into the Introduction section and the Method section. The author may move the background information and TF classification to the Introduction section, and include an Implementation section in the Methods section to describe how TFutils incorporates all these data sources and how to retrieve the relevant information in TFutils and how to integrate with other packages as mentioned in 2, where the R script snippets can be displayed. To maintain/increase the user base, it is important to keep the data up to date. Currently, the data were snapshots of the CISBP and HOCOMOCO catalogs. If the resources are not updated regularly, it’s unlikely that users will use TFutils after 2-3 years. Is there a plan in place to have the resources assembled by TFutils be update regularly? How often is the update going to be? Is it going to be automatic or manually? Flexibility of the data structure is also important, as users may want to expand the utility of TFutils. Suggest authors describe how to add features to the current data structures in TFutils in the manuscript. It will be useful to add information on the numbers of TFs and targets included in the assembled resources, as well as in the original databases. There is a python package having the same name “tfutils” which is very popular. If it is not too hard to do, we suggest authors change the package name to avoid confusion Installation and running environments of the TFutils was described twice, once in the Introduction section, the other time in the Methods section: Operation: Installation. It is better to only describe this once in the Methods section. There are many short paragraphs consisting of one or two sentences and related information are scattered into different sections. For instances, the last paragraph of the Introduction section about the limitations of TFutil might be moved to the Discussion part; whereas the third paragraph in the Discussion section might be moved to somewhere at the beginning of the Introduction section or where it is appropriate. Page 6, the Summary section might be better moved to between the data availability section and the discussion section to summarize the implemented functionality of TFutil.\nBesides those major issues, we also have a few minor questions:\nCurrently the abstract only mentions TF targets derived from the MSigDb. Considering that the ENCODE TF ChIP-seq data is one of the most significant resources for TF targets information as mentioned in the main text, suggest authors add how the ENCODE ChIP-seq data were incorporated into TFutils in the abstract. Page 5, please clarify the type of details in the sentence “Full details are provided in Sonawane et al”. Gene structure can be better depicted in Fig. 3 and Fig. 4, perhaps adopting the gene structure visualization in most genome viewers, showing exon/intron structure and gene transcription direction. Please include the used R packages in the citation. “TFtargs()” in Figure 5 legend needs to be edited. For the subtitles under the Use cases section, suggest add find before “TFs that are direct GWAS …” and retrieve before “Traits mapped to genes that …”.\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": "4634", "date": "17 May 2019", "name": "Vincent Carey", "role": "Author Response", "response": "We appreciate the careful reading of this paper, which has inspired additional functionality that will be added to the package. Reviewer comments (isolated with QUERY: tag) are addressed below (with RESPONSE: tag).QUERY: TFutils includes resources from CISBP, HOCOMOCO, GO and MSigDb. There are additional humanTF resources. Is there any reason not to include those resources such as JASPAR1, Transfac2, HDPI3 and uniPro4?RESPONSE: According to http://cisbp.ccbr.utoronto.ca/summary.php?by=4&orderby=MSource_Identifier JASPAR and TRANSFAC entries are included in CIS-BP. UniProbe is an interesting resource but does not provide a mapping from TF or motif to target genes. Interfacing to UniProbe is beyond the scope of the tasks intended for this paper. The same issue appears to us to apply to hPDI. Resources that do not enumerate \"gene level\" TF targets are beyond the scope of current work.QUERY: There are potential packages that will likely import TFutils such as TFBSTools for the analysis of transcription factor binding sites manipulation, motifStack for graphic representation of multiple motifs5 and MotIV. It will be helpful to present a few lines of code to show how to integrate data from TFutils to aforementioned pipelines.RESPONSE: We will add an example of how diversity in TF reference motif data leadsto a structure that may be visualized with MotifDb/motifStack. Specifically the 1.5.x+ versions of TFutils will include a function that uses motifStack and MotifDb to generate displays like Figure 5 of the revised paper. This is the result of example(tffamCirc.plot)in the current devel branch of TFutils.QUERY: The section “Basic concepts of transcription factor bioinformatics” includes lots of background information, such as existing TF-related data sources/bases, TF classification, and how TFutils incorporates and access those resources. To make it easy to follow, we suggest break this part into the Introduction section and the Method section. The author may move the background information and TF classification to the Introduction section, and include an Implementation section in the Methods section to describe how TFutils incorporates all these data sources and how to retrieve the relevant information in TFutils and how to integrate with other packages as mentioned in 2, where the R script snippets can be displayed.RESPONSE: Some of these changes are made. Our approach to the narrative attempts to follow the F1000Research schema.QUERY: To maintain/increase the user base, it is important to keep the data up to date. Currently, the data were snapshots of the CISBP and HOCOMOCO catalogs. If the resources are not updated regularly, it’s unlikely that users will use TFutils after 2-3 years. Is there a plan in place to have the resources assembled by TFutils be update regularly? How often is the update going to be? Is it going to be automatic or manually?RESPONSE: The package will be updated according to the Bioconductor releaseprotocol. The main pages give explicit information on provenance of information underlying serialized data structures. Community input on the utility of the various sources will be important in determining the frequency of content updates.QUERY: Flexibility of the data structure is also important, as users may want to expand the utility of TFutils. Suggest authors describe how to add features to the current data structures in TFutils in the manuscript.RESPONSE: The code is open source. Pull requests are welcome. If there are specific features of interest to the reviewers we will consider how to incorporate them in future versions of the package/manuscript.QUERY: It will be useful to add information on the numbers of TFs and targets included in the assembled resources, as well as in the original databases.RESPONSE: Totals are added in the caption of Figure 1.QUERY: There is a python package having the same name “tfutils” which is very popular. If it is not too hard to do, we suggest authors change the package name to avoid confusionRESPONSE: The python package addresses \"tensorflow\", which is not related to TFsin bioinformatics. We do not believe that the risk of confusion is high, but will consider renaming if events of confusion are observed.QUERY: Installation and running environments of the TFutils was described twice, once in the Introduction section, the other time in the Methods section: Operation: Installation. It is better to only describe this once in the Methods section.RESPONSE: The presentation follows the suggested F1000Research format. QUERY: There are many short paragraphs consisting of one or two sentences and related information are scattered into different sections. For instances, the last paragraph of the Introduction section about the limitations of TFutil might be moved to the Discussion part; whereas the third paragraph in the Discussion section might be moved to somewhere at the beginning of the Introduction section or where it is appropriate.Page 6, the Summary section might be better moved to between the data availability section and the discussion section to summarize the implemented functionality of TFutil.RESPONSE: The last paragraph of the introduction is used to pre-empt potential reader disappointment early in the presentation, so we prefer to leave it where it is. The third paragraph in the discussion does include content of general and possibly introductory significance, but that content is embedded in concrete illustrations that depend upon actual details of package use. The \"summary\" element is provided to give the reader a break before plunging into the obligatory Methods/Implementation material. We concur with your basic aesthetic preferences but have organized our text in what we consider to be a rational way.QUERY: Besides those major issues, we also have a few minor questions:QUERY: Currently the abstract only mentions TF targets derived from the MSigDb. Considering that the ENCODE TF ChIP-seq data is one of the most significant resources for TF targets information as mentioned in the main text, suggest authors add how the ENCODE ChIP-seq data were incorporated into TFutils in the abstract.RESPONSE: metadata(encode690) provides details. The ENCODE data are derived fromBioconductor's AnnotationHub package. These facts are noted in the vicinity of Figure 4. We have added a phrase to the abstract that mentions the ENCODE interface.QUERY: Page 5, please clarify the type of details in the sentence “Full details are provided in Sonawane et al”.RESPONSE: These authors describe how FIMO was used to obtain sequence-based binding affinity scores; the main text is slightly modified to clarify the role of this reference.QUERY: Gene structure can be better depicted in Fig. 3 and Fig. 4, perhaps adopting the gene structure visualization in most genome viewers, showing exon/intron structure and gene transcription direction.RESPONSE: We agree that the gene model displays are sub-optimal. However, the visualizations are not central to the package. Ideally, Gviz, ggbio, or karyploteR infrastructure would be used and we will pursue these improvements in updates to the package. Further discussion of the visualization is conducted with the other referee report. We note that the interactive \"app\" at https://vjcitn.shinyapps.io/encdemo/ can be used to interactively view binding sites for a small number of TFs in a small number of cell types. Such visualizations can be better accomplished with standard genome browsers. The visualizations in the paper are provided to make concrete the immediate programmatic availability of these resources and concepts to package users. In particular, Figure 3 just scratches the surface of the concept that the combinatorics of TF binding are complex. As noted by the other reviewer, the display is impossible to parse meaningfully in detail, but the overall interpretation is that binding events form a complex ensemble and that data structures and programming patterns are needed to develop compelling interpretations.QUERY: Please include the used R packages in the citation.RESPONSE: After running the Rmarkdown document in TFutils/vignette/TFutils_f1000 that conducts all the computations presented in the paper, sessionInfo() can be run to enumerate all packages in use. There are 37 packages attached, and 60 loaded but not attached. It does not seem reasonable to burden the paper with such an accounting. Users can run sessionInfo() and then query the DESCRIPTION files of packages of interest for provenance information.QUERY: “TFtargs()” in Figure 5 legend needs to be edited.RESPONSE: Done.QUERY: For the subtitles under the Use cases section, suggest add find before “TFs that are direct GWAS …” and retrieve before “Traits mapped to genes that …”.RESPONSE: Done." } ] }, { "id": "45003", "date": "26 Apr 2019", "name": "Giovanna Ambrosini", "expertise": [ "Reviewer Expertise Bioinformatics", "Epigenetics", "ChIP-seq", "regulatory region annotation", "motif analysis", "database design", "web tools." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTFutils is a Bioconductor package meant to help users study TF binding in the human genome. The tool integrates several resources such as Gene Ontology (GO), CISBP, HOCOMOCO, and MSigD (the Molecular Signature Database). The paper describes how to tackle basic problems users are faced with when trying to work with TFs, in particular the TF classification, the gene targets identification, and, ultimately, the prediction of TF binding affinities.\n\nThis article looks more like a software tutorial than a scientific article. As software has to evolve in order keep up with user needs, the text will have to be updated on a regular basis in the future, in order to remain up-to-data as well. This is fine if F1000Research accepts updates and supports versioning of articles. Otherwise, another format should be chosen for presenting this tool.\n\nJust by reading the article, we didn't get a clear impression of what is inside TFutils. Going through the command examples was helpful in this respect. Nevertheless, we have doubts whether we would be able to use this package in a productive manner in the future. The promise that \"TFutils lowers the barriers of usage of key findings of TF biology\" holds only for expert users of Bioconductor, who are already familiar with all the other packages mentioned in this article and necessary to reproduce the results.\n\nThe current manuscript has several shortcomings. At a general level, it is not very transparent to the naïve reader what is actually new from this package and what functionalities are provided by the many other Bioconductor packages referred to in the text. Fortunately, we found a well-organized reference manual for TFutils version 1.2.0 on the internet, which clarified this issue for us. A URL to this document should have been included in the article.\n\nAs this is a tutorial-style document, it would be helpful to provide complete R code for reproducing Figures 3 and 4. While Figure 3 is relatively easy to generate, it took us at least half a day to reproduce Figure 4. A major limitation is that the fimo16 object, upon which Figure 3 is based, contains only TF affinity data for 16 out of 689 scanned TF motif matrices.\n\nFigure 3 shows the predicted binding sites for 16 TFs in a selected genomic region. Already with such a small number of TFs, the Figure is pretty crowded with dots. One wonders what it would look like if all 689 FIMO-scanned motif matrices were considered. In view of the density of motif matches it seems doubtful whether any biological insights can be gained from such a plot. Some guidance for the interpretation is needed.\n\nFigure 4 shows ENCODE binding peaks for CEBPB in the same genomic region that was used for Figure 3. Naturally, we were curious to know whether the peaks seen in this Figures co-localize with corresponding motif matches in Figure 3. Unfortunately, CEBPB is not included in the fimo16 collection. To exemplify the power of the tool, it would have been preferable to choose an example where the reader can crosscheck the consistency between predictions and experiments via comparison of Figure 3 with Figure 4.\n\nOverall, our impression is that TFutils is a useful package albeit for a restricted community of users already familiar with the other Bioconductor packages mentioned in the article. However, the manuscript could benefit from major revisions as pointed out 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? 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": "4635", "date": "17 May 2019", "name": "Vincent Carey", "role": "Author Response", "response": "We are very appreciative of the effort underlying this review. Important questions were raised and we endeavor to answer them fully below. Reviewer comments are prefaced by \"QUERY\" and our replies are prefaced by \"RESPONSE\".QUERY: This article looks more like a software tutorial than a scientific article. As software has to evolve in order keep up with user needs, the text will have to be updated on a regular basis in the future, in order to remain up-to-data as well. This is fine if F1000Research accepts updates and supports versioning of articles. Otherwise, another format should be chosen for presenting this tool.RESPONSE: Because F1000Research accepts updates and supports versioning of articles, we believe that this format is an acceptable one for the work we have described.QUERY: Just by reading the article, we didn't get a clear impression of what is inside TFutils. Going through the command examples was helpful in this respect. Nevertheless, we have doubts whether we would be able to use this package in a productive manner in the future. The promise that \"TFutils lowers the barriers of usage of key findings of TF biology\" holds only for expert users of Bioconductor, who are already familiar with all the other packages mentioned in this article and necessary to reproduce the results.RESPONSE: We are glad that the examples in the paper were useful to the reviewer. It is true that Bioconductor software generally requires acquaintance with and use of multiple interrelated packages. In this sense, it is different from a relatively common approach of command-line utility implementation of bioinformatic analysis tools, where a given tool may be understood and used in isolation. We do not agree that \"expert\" level of understanding of Bioconductor is necessary to make use of the material described, although some facility with and enthusiasm for the R language would be necessary to make headway. The paper is published in the Bioconductor channel of F1000research, and it is expected that the readership will have an acquaintance with the resources and limitations of the Bioconductor software and data ecosystem.QUERY: The current manuscript has several shortcomings. At a general level, it is not very transparent to the naïve reader what is actually new from this package and what functionalities are provided by the many other Bioconductor packages referred to in the text. Fortunately, we found a well-organized reference manual for TFutils version 1.2.0 on the internet, which clarified this issue for us. A URL to this document should have been included in the article.RESPONSE: This is a useful observation. We have now included a reference to http://bioconductor.org/packages/release/bioc/manuals/TFutils/man/TFutils.pdfin the implementation section of the paper.QUERY: As this is a tutorial-style document, it would be helpful to provide complete R code for reproducing Figures 3 and 4. While Figure 3 is relatively easy to generate, it took us at least half a day to reproduce Figure 4. A major limitation is that the fimo16 object, upon which Figure 3 is based, contains only TF affinity data for 16 out of 689 scanned TF motif matrices.RESPONSE: We appreciate the effort taken here. The production of Figure 4 was complicated and we have created an app and associated github repository to clarify the basic issues. The repository is https://github.com/vjcitn/encdemo and the face page of the repo has a screenshot of the app, which runs at https://vjcitn.shinyapps.io/encdemo/. Our point in the visualization of Figure 4 is not the visualization per se, which can be accomplished with standard genome browsers, with suitable commands. Rather, we use the visualization to give concrete demonstration of the immediate programmatic availability (to users of this package) of the relevant experimental results and annotations. We concur that the limitation of fimo16 to a small number of TFs is disappointing. Comprehensive presentation of the scan scores to our user base/readership would require computational resources that we have not yet been able to muster. A local deployment requires close to a terabyte of indexed storage.QUERY: Figure 3 shows the predicted binding sites for 16 TFs in a selected genomic region. Already with such a small number of TFs, the Figure is pretty crowded with dots. One wonders what it would look like if all 689 FIMO-scanned motif matrices were considered. In view of the density of motif matches it seems doubtful whether any biological insights can be gained from such a plot. Some guidance for the interpretation is needed.RESPONSE: As noted just above we do not have a mechanism for providing all 689 scans. We have added text after Figure 3 acknowledging the challenge of interpretation, specifically with respect to combinatorics of TF binding.QUERY: Figure 4 shows ENCODE binding peaks for CEBPB in the same genomic region that was used for Figure 3. Naturally, we were curious to know whether the peaks seen in this Figures co-localize with corresponding motif matches in Figure 3. Unfortunately, CEBPB is not included in the fimo16 collection. To exemplify the power of the tool, it would have been preferable to choose an example where the reader can crosscheck the consistency between predictions and experiments via comparison of Figure 3 with Figure 4.RESPONSE: We agree that unification of concepts underlying Figure 3 and 4 would be quite desirable. Figure 3 is based on the analysis of motifs in reference sequence, while Figure 4 is a severe reduction of cell-type specific information from in vitro experiments. The data underlying the encdemo app noted above should be useful for beginning surveys across TFs and across cell types essential for a full understanding of cell-type specific combinatorics of TF binding.QUERY: Overall, our impression is that TFutils is a useful package albeit for a restricted community of users already familiar with the other Bioconductor packages mentioned in the article. However, the manuscript could benefit from major revisions as pointed out above.RESPONSE: We appreciate the effort taken in this review and we have endeavored to answer the questions raised." } ] } ]	1	https://f1000research.com/articles/8-152
https://f1000research.com/articles/8-672/v1	16 May 19	{ "type": "Research Article", "title": "The impact of physical activity to the child’s quality of life: a bibliometric study", "authors": [ "Jernej Završnik", "Peter Kokol", "Helena Blažun Vošner", "Peter Kokol", "Helena Blažun Vošner" ], "abstract": "Background: The application of bibliometrics in healthcare research is becoming popular, however at present it is still an under-researched area. Methods: In our study we used a bibliometric technique called bibliometric mapping to visualize the published research regarding the influence of physical activity to children’s quality of life. The research was visualized in the form of both chronological and cluster science landscapes. Science landscapes, contrary to conventional reviews, capture the relationships between multiple topics and concepts, enabling the generation of “synthetic reviews”. Results: Evolutionarily, three distinct research phases appeared, namely research on influence of physical activity on various chronic non-communicable diseases; research on quality of life and childhood diseases related to physical activity; and outcome-related research. The research consists of six main topics: asthmatic child and exercising, blood diseases, health-related quality of life, obesity and chronic diseases, childhood obesity and behaviour, and depression and health outcomes. Conclusions: The study identified some research that may be helpful to general paediatricians whose everyday practice or research is not focused on physical activity and child’s quality of life, but wants to learn about the taxonomy of the topics, the most interesting discoveries, guidelines and practices and the state of the art in the field. It also revealed some hidden association, otherwise not easily identified, even by informed researchers and clinicians.", "keywords": [ "Physical activity", "Health related quality of life", "Bibliometric mapping", "Scientific landscape" ], "content": "Introduction\n\nThe role of bibliometrics in healthcare research has been excellently described by Lewison and Devey1: “Bibliometrics is to scientific papers as epidemiology is to patients.” Indeed, in 1990 bibliometrics first became a medical subject heading. Consequently, the application of bibliometrics by health professionals to analytical, clinical, informational and academic areas of interest is becoming more extensive; however, at present it is an unexploited area of a fruitful research2.\n\nInterdisciplinary research concerning the influence of physical activity and sport on children’s quality of life is increasingly becoming more and more important. In their review paper Buttitta et al.3 reported that several factors are associated with children’s quality of life; among them physical activity is one of the most important, especially in in those with pre-existing diseases. Various malignant diseases and anticancer therapy in children both drastically affect daily life activities, including high-performance sports. However, both random and non-random feasibility studies show positive effects of physical activity on clinical and psychosocial outcomes. Consequently, every effort should be made to maintain physical activity during paediatric cancer therapy4. Comparable findings for congenital heart diseases, were reported by Dulfer et al.5.\n\nSince our research area interest is multidisciplinary, we had an opportunity to employ a bibliometric technique called bibliometric mapping. Bibliometric mapping visualizes academic research in the form of scientific landscapes6. Science landscapes, contrary to conventional reviews, which focus on particular research questions, extract information at various levels and capture the relationship between multiple topics and concepts, creating “synthetic reviews”. Scientific landscapes are still relatively rarely used; however, those published have been well received by the scientific community7.\n\n\nMethods\n\nBibliometrics could be defined as the application of mathematical, statistical and heuristic methods to scientific publications8. Bibliometric mapping is a recent addition to techniques already used in bibliometrics in medicine2. Bibliometric mapping aims to visualize different facets of literature production based on different co-occurrences (i.e. words, authors, organisations, journals)9, co-citations10 and bibliographical coupling11. Bibliometric mapping can be automated using various software tools. Among open licence software tools, VOSviewer12 is very versatile and easy to use13. Both bibliometric mapping and VOSViewer have been successfully used in health related fields14.\n\nThe corpus was extracted from the Scopus bibliographical dataset, because of Scopus broad coverage of various journals, book chapters and conference proceedings on one hand and on the other hand because of Scopus’ extensive and easy to use search and analytical functions. The search was made on 12th January 2019 using the search keyword string child* AND (\"physical activity\" or \"sport\") AND \"quality of life\" in information source titles, abstracts and keywords for the whole period covered by Scopus.\n\nPublication year, source title and author's country of affiliation were extracted by Scopus services. Abstracts were exported as comma separated values (CSV) files to enable further analysis by the VOSviewer (V1.6.9) and Excel software (Version 2016). Cluster and chronological scientific landscapes were generated for all terms with an occurrence frequency larger than 30. Common words such as report, significance, trial, study and baseline were ignored using a customised thesaurus file (see Underlying data15). Similarly, synonyms like body mass index and BMI were integrated into one term. The Attraction was set to 4, Min. Cluster size to 8, Resolution to 1.25; all other VOSViewer parameters were set to default values.\n\nThe popularity of a term (size of the term font and associated square) and relatedness between terms (terms located near each other are more related than those further apart) in both cluster and chronological scientific landscapes were analysed using meta-synthesis16. An appropriate topic was determined for each cluster based on the analysis of most popular terms belonging to the cluster.\n\n\nResults\n\nThe search resulted in 2334 publications (1637 articles, 419 reviews, 66 conference papers, 50 editorials, 35 notes, 29 book chapters, 19 letters, 19 short communications and 24 other types of publications; see Underlying data15). The most popular journals in which the above publications appeared are shown in Table 1. The most prolific journal was BMC Public Health with 31 publications. The highest ranked journal in the selected field was Pediatrics, with 21 publications (621st place according to SCImago Journal Rank - SJR, which ranks within the top 2% of all source titles indexed by SciMago. Interestingly, all of the most prolific source titles are ranked in the top quarter of all journals. Their contribution to the total literature production on our topic of interest accounts for 12.4%. As expected most of the most prolific source titles are from the field of paediatrics, others relate to quality of life or from general areas. Surprisingly no source titles related to sport or physical activity were ranked among the most prolific source titles – the first (American Journal of Sports Medicine) is in 15th place with 11 publications.\n\nThe most productive countries are presented in Table 2. All of them have advanced heath, industrial and economic systems, high BDP and are also leading countries in research and development. The top 10 countries produced more than 83% of all publications, indicating that literature production is regionally centred.\n\nShen et al.17 proposed a model of science discipline development based on literature production dynamics. Following their example, a graph was constructed (Figure 1) which reveals three distinguished phases in the production of publications regarding influence of physical activity to children’s quality of life, namely:\n\ninitial phase in the period 1975–1989 when publications were scare, most three publications per year;\n\ninitiation phase in the period 1990–1999 when number of publications linearly increased from 6 to 23; and\n\nexponential growth phase in the period 2000–2018, when production reached its peak in 2017 with 231 publications.\n\nFigure 2 presents the term map on the topic of research, the influence of physical activity to children’s quality of life. According to the figure the research development went through three main phases, namely:\n\n1. Research on the influence of physical activity on non-communicable diseases (approx. period from 1975 to 2011 – violet and blue colours). The associations between (1) hypertension, diabetes, cardiovascular diseases, mortality, exercise and positive/beneficial effects, and (2) asthma, cystic fibrosis, medication, illness, sport and daily activity, were the main stream of research in the first phase.\n\n2. Research on frequent childhood diseases and physical activity (green and light blue colours – approx. period from 2012 to 2013). The research was focused on associations between (1) chronic diseases, society, pregnancy, birth, sedentary life style, prevention strategies, and risk increase (2), nutrition, promotion, blood pressure, obesity, physical activity and, fitness and (3) anxiety, depression, stress, juvenile idiopathic arthritis, haemophilia, injury and health status.\n\n3. Measuring quality of life (light green and yellow colours – state-of-the-art research). The research in the most recent period is concerned with association between (1) inclusion, adherence, protocol, cancer and health outcomes, (3) sedentary behaviour, weight status, physical activity level and physical activity interventions and (4) quality of life indicators, physical functioning and physical health.\n\nThe cluster science landscape (Figure 3) consists of six clusters. Meta-synthesis revealed six topics, as shown in Table 3.\n\n\nDiscussion\n\nIt is interesting to note some details evident from the derived topics above and associations between terms:\n\nThe quantity of research concerning physical activity in relation to asthma, but not only as a single disease, but also in combination with obesity and diabetes. Di Genova et al.18 claim that growing evidence shows the existence of an “obese asthma” phenotype characterised by difficult-to-manage asthma. Additionally, Atay and Berket19 show that obesity results in various co-morbidities in children and adolescent.\n\nThe increasing incidence of psychosomatic diseases and the beneficial effects of physical activity. Hrafnkelsdottir et al.20 reports that less screen time and more intense physical activity lowers the risk of mental health problems.\n\nThe broadness of the spectrum of diseases related to child’s quality of life and physical activity. Studies highlighted that children with overweight/obesity and related co-morbidities and lifestyle behavioural risk factors, had significantly lower healthy-related quality of life21,22.\n\nThe introduction of modern technology such as various sensors in both research and practice supporting the empirical research in areas that were previously very subjective. Traffic-related air pollution and noise may lead to adverse health outcomes, including increased blood pressure, myocardial infarction, and respiratory health in paediatric population. Measuring the physical activity and environmental factors revealed that such measurements can lead to better understanding of the relation between above factors23. Another study investigated the use of a carbohydrate intake based on continuous glucose monitoring trends during physical activity of children with diabetes to objectively asses the association between these two variables24.\n\nThe state-of-the-art efforts in the development of specific child’s quality of life indices and instruments to measure their relatedness to other paediatric indices. Moghaddaszadeh et al.25 measured the paediatric quality of life indicators and their relations to enjoyment levels and physical attractiveness.\n\nThe relationship between acute lymphoblastic leukaemia and haemophilia, and physical activity. Acute lymphoblastic leukaemia treatment in children can result in muscle weakness and motion limitations. A study showed that active dorsiflexion range of motion combined with physical activity had a positive correlation with strength/agility standard score26. Haemophilia management recommends physical activity in children with haemophilia. In a study researchers adapted and validated the adult Haemophilia & Exercise Project-Test-Questionnaire (HEP-Test-Q) for children aged 6–17 years, reformulated questionnaire items to make them understandable to children27.\n\n\nConclusion\n\nUsing bibliometric mapping we created two scientific landscapes on the topic of research, the influence of physical activity to children’s quality of life. To the best of our knowledge, this is the first such attempt in the paediatric field. We identified six distinct topics and also visualised the chronological aspect of the research literature production. The study revealed some knowledge that might be helpful to an “outsider” who wants to learn about the taxonomy of the topics, the most interesting discoveries, guidelines and practices and the state of the art in the field. It can also help the “seasoned insiders” better understand more specialised research including that is out of their immediate scope of interest. Additionally, it can reveal hidden facts, not easily identified, even by informed researchers and clinicians.\n\n\nData availability\n\nOpen Science Framework: Physical activity and quality of life in children. https://doi.org/10.17605/OSF.IO/WXSQE15\n\nThis project contains the following underlying data:\n\nMap.txt (Map file for VOSViewer software)\n\nNetwork.txt (Network file for VOSViewer software)\n\nScopus1.csv (Extracted articles from Scopus 2011–2019)\n\nScopus2.csv (Extracted articles from Scopus 1975–2010)\n\nThesaurus_terms.txt (Customised thesaurus file defining terms to be omitted and synonyms)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLewison G, Devey ME: Bibliometric methods for the evaluation of arthritis research. Rheumatology (Oxford). 1999; 38(1): 13–20. PubMed Abstract \| Publisher Full Text\n\nAlfonzo PM, Sakraida TJ, Hastings-Tolsma M: Bibliometrics: Visualizing the impact of nursing research. Online J Nurs Inform. 2014; 18(1). Reference Source\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): 1117–39. PubMed Abstract \| Publisher Full Text\n\nGötte M, Taraks S, Boos J: Sports in pediatric oncology: the role(s) of physical activity for children with cancer. J Pediatr Hematol Oncol. 2014; 36(2): 85–90. PubMed Abstract \| Publisher Full Text\n\nDulfer K, Helbing WA, Duppen N, et al.: Associations between exercise capacity, physical activity, and psychosocial functioning in children with congenital heart disease: a systematic review. Eur J Prev Cardiol. 2014; 21(10): 1200–15. PubMed Abstract \| Publisher Full Text\n\nKokol P, Blažun Vošner H, Železnik D: Clinical Simulation in Nursing: A Bibliometric Analysis after Its Tenth Anniversary. Clin Simul Nurs. 2017; 13(4): 161–7. Publisher Full Text\n\nLee CISG, Felps W, Baruch Y: Toward a taxonomy of career studies through bibliometric visualization. J Vocat Behav. 2014; 85(3): 339–51. Publisher Full Text\n\nBellis ND: Bibliometrics and Citation Analysis: From the Science Citation Index to Cybermetrics. Lanham, Md: Scarecrow Press; 2009; 450. Reference Source\n\nChen X, Chen J, Wu D, et al.: Mapping the Research Trends by Co-word Analysis Based on Keywords from Funded Project. Procedia Comput Sci. 2016; 91: 547–55. Publisher Full Text\n\nSmall H: Co-citation in the scientific literature: A new measure of the relationship between two documents. J Am Soc Inf Sci. Wiley Online Library [Internet]. [cited 2019 Apr 5]. 1973; 24(4): 265–269. Publisher Full Text\n\nBiscaro C, Giupponi C: Co-authorship and bibliographic coupling network effects on citations. PLoS One. 2014; 9(6): e99502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Eck NJ, Waltman L, Noyons EC, et al.: Automatic term identification for bibliometric mapping. Scientometrics. 2010; 82(3): 581–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlažun H, Kokol P, Vošner J: Research literature production on nursing competences from 1981 till 2012: A bibliometric snapshot. Nurse Educ Today. 2015; 35(5): 673–9. PubMed Abstract \| Publisher Full Text\n\nKokol P, Završnik J, Blažun Vošner H: Bibliographic-Based Identification of Hot Future Research Topics: An Opportunity for Hospital Librarianship. J Hosp Librariansh. 2018; 18(4): 315–322. Publisher Full Text\n\nKokol P: Physical activity and quality of life in children. 2019. http://www.doi.org/10.17605/OSF.IO/WXSQE\n\nBondas T, Hall EO: Challenges in approaching metasynthesis research. Qual Health Res. 2007; 17(1): 113–21. PubMed Abstract \| Publisher Full Text\n\nShen J, Yao L, Li Y, et al.: Visualizing the history of evidence‐based medicine: A bibliometric analysis. J Am Soc Inf Sci Technol. Wiley Online Library [Internet]. [cited 2019 Apr 5]. 2013; 64(10): 2157–2172. Publisher Full Text\n\nDi Genova L, Penta L, Biscarini A, et al.: Children with Obesity and Asthma: Which Are the Best Options for Their Management? Nutrients. 2018; 10(11): pii: E1634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtay Z, Bereket A: Current status on obesity in childhood and adolescence: Prevalence, etiology, co-morbidities and management. Obes Med. 2016; 3: 1–9. Publisher Full Text\n\nHrafnkelsdottir SM, Brychta RJ, Rognvaldsdottir V, et al.: Less screen time and more frequent vigorous physical activity is associated with lower risk of reporting negative mental health symptoms among Icelandic adolescents. PLoS One. 2018; 13(4): e0196286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoare E, Crooks N, Hayward J, et al.: Associations between combined overweight and obesity, lifestyle behavioural risk and quality of life among Australian regional school children: baseline findings of the Goulburn Valley health behaviours monitoring study. Health Qual Life Outcomes. 2019; 17(1): 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuen KC, Koltowska-Häggström M, Cook DM, et al.: Clinical characteristics and effects of GH replacement therapy in adults with childhood-onset craniopharyngioma compared with those in adults with other causes of childhood-onset hypothalamic-pituitary dysfunction. Eur J Endocrinol. 2013; 169(4): 511–9. PubMed Abstract \| Publisher Full Text\n\nLeaffer D, Wolfe C, Doroff S, et al.: Wearable Ultrafine Particle and Noise Monitoring Sensors Jointly Measure Personal Co-Exposures in a Pediatric Population. Int J Environ Res Public Health. 2019; 16(3): pii: E308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurckhardt MA, Chetty T, Smith GJ, et al.: Use of Continuous Glucose Monitoring Trends to Facilitate Exercise in Children with Type 1 Diabetes. Diabetes Technol Ther. 2019; 21(1): 51–5. PubMed Abstract \| Publisher Full Text\n\nMoghaddaszadeh A, Jamnik V, Belcastro AN: Characteristics of children’s physical activity during active play. J Sports Med Phys Fitness. 2018; 58(4): 369–76. PubMed Abstract \| Publisher Full Text\n\nTanner LR, Hooke MC: Improving body function and minimizing activity limitations in pediatric leukemia survivors: The lasting impact of the Stoplight Program. Pediatr Blood Cancer. 2019; 66(5): e27596. PubMed Abstract \| Publisher Full Text\n\nvon Mackensen S, Hilberg T, Valentino LA, et al.: Validation of the Haemophilia & Exercise Project-Test-Questionnaire (HEP-Test-Q)-An instrument for the assessment of subjective physical functioning in children with haemophilia. Haemophilia. 2018; 24(6): 888–95. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49710", "date": "17 Jun 2019", "name": "Zachi Grossman", "expertise": [ "Reviewer Expertise General paediatrics" ], "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 find this manuscript interesting since it gives a new perspective on the literature.\nI would like to propose the following changes in order to improve the conclusions:\nSingle papers on blood diseases - haemophilia and leukaemia - are not enough to draw conclusions from. Therefore, I would tend to disregard these papers both in the discussion and in the abstract. I am not so convinced in the existence of three distinguished time phases. I would prefer a two time phases division: before and after 1995.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "50557", "date": "25 Jul 2019", "name": "Manuel Jesus Cobo", "expertise": [ "Reviewer Expertise Bibliometric and science mapping 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 manuscript develop a science mapping analysis of the physical activity on children and the effect in their quality of life. To do that, VOSViewer software tool is employed.\nIn general, the paper is well organised and written. Moreover, the software tool is appropriate for this analysis and authors use it properly.\nIn what follows, some comments and suggestions are listed:\nI miss some references to the origins of co-word analysis, science mapping, or generally, science of science. Authors focus too much on bibliometrics on health, but there are important references out of this field. Moreover, authors should cite some recent bibliometric studies developed in health, for example in cancer, rehabilitations, etc.\n\nThe reference to VOSViewer is incorrect. Author should cite the paper where the software is presented.\n\nFigure 2 is confusing. Authors should provide other kind of visualisation. Also, authors should describe better the conceptual evolution of this research field.\n\nDiscussion should be better described. At this moments it is just a sequence of itemises. Author should provided a clear speech.\n\nConclusions are just a summary of the paper.\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? Yes", "responses": [] }, { "id": "52912", "date": "02 Sep 2019", "name": "Julije Mestrovic", "expertise": [ "Reviewer Expertise Paediatric intensive care and quality of life." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 work is interesting for readers and should be indexed. The following should be done first:\nIn the discussion, explain briefly the possible relationships between illness and physical activity. Namely, it is important to note what are the mechanisms of how physical activity can affect disease, but also vice versa, in a wide range of conditions, from asthma to air pollution.\n\nIn discussion, avoid overlapping conditions (asthma and obesity).\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": [] } ]	1	https://f1000research.com/articles/8-672
https://f1000research.com/articles/8-338/v1	26 Mar 19	{ "type": "Research Article", "title": "Are we missing ‘previously treated’ smear-positive pulmonary tuberculosis under programme settings in India? A cross-sectional study", "authors": [ "Hemant Deepak Shewade", "Vivek Gupta", "Srinath Satyanarayana", "Atul Kharate", "Lakshmi Murali", "Madhav Deshpande", "Naresh Kumar", "Prabhat Pandey", "U N Bajpai", "Jaya Prasad Tripathy", "Soundappan Kathirvel", "Sripriya Pandurangan", "Subrat Mohanty", "Vaibhav Haribhau Ghule", "Karuna D Sagili", "Banuru Muralidhara Prasad", "Sudhi Nath", "Priyanka Singh", "Kamlesh Singh", "Gurukartick Jayaraman", "P Rajeswaran", "Binod Kumar Srivastava", "Moumita Biswas", "Gayadhar Mallick", "Om Prakash Bera", "A James Jeyakumar Jaisingh", "Ali Jafar Naqvi", "Prafulla Verma", "Mohammed Salauddin Ansari", "Prafulla C Mishra", "G Sumesh", "Sanjeeb Barik", "Vijesh Mathew", "Manas Ranjan Singh Lohar", "Chandrashekhar S Gaurkhede", "Ganesh Parate", "Sharifa Yasin Bale", "Ishwar Koli", "Ashwin Kumar Bharadwaj", "G Venkatraman", "K Sathiyanarayanan", "Jinesh Lal", "Ashwini Kumar Sharma", "Ajay MV Kumar", "Sarabjit S Chadha", "Vivek Gupta", "Srinath Satyanarayana", "Atul Kharate", "Lakshmi Murali", "Madhav Deshpande", "Naresh Kumar", "Prabhat Pandey", "U N Bajpai", "Jaya Prasad Tripathy", "Soundappan Kathirvel", "Sripriya Pandurangan", "Subrat Mohanty", "Vaibhav Haribhau Ghule", "Karuna D Sagili", "Banuru Muralidhara Prasad", "Sudhi Nath", "Priyanka Singh", "Kamlesh Singh", "Gurukartick Jayaraman", "P Rajeswaran", "Binod Kumar Srivastava", "Moumita Biswas", "Gayadhar Mallick", "Om Prakash Bera", "A James Jeyakumar Jaisingh", "Ali Jafar Naqvi", "Prafulla Verma", "Mohammed Salauddin Ansari", "Prafulla C Mishra", "G Sumesh", "Sanjeeb Barik", "Vijesh Mathew", "Manas Ranjan Singh Lohar", "Chandrashekhar S Gaurkhede", "Ganesh Parate", "Sharifa Yasin Bale", "Ishwar Koli", "Ashwin Kumar Bharadwaj", "G Venkatraman", "K Sathiyanarayanan", "Jinesh Lal", "Ashwini Kumar Sharma", "Ajay MV Kumar", "Sarabjit S Chadha" ], "abstract": "Background: In 2007, a field observation from India reported 11% misclassification among ‘new’ patients registered under the revised national tuberculosis (TB) control programme. Ten years down the line, it is important to know what proportion of newly registered patients has a past history of TB treatment. Methods: A study was conducted among new smear-positive pulmonary TB patients registered between March 2016 and February 2017 in 18 randomly selected districts to determine the effectiveness of an active case-finding strategy in marginalised and vulnerable populations. We included all patients detected through active case-finding. An equal number of randomly selected patients registered through passive case-finding from marginalised and vulnerable populations in the same districts were included. Before enrolment, we enquired about any history of previous TB treatment through interviews. Results: Of 629 patients, we interviewed 521, of whom, 11% (n=56) had past history of TB treatment (public or private) for at least a month: 13% (34/268) among the active case-finding group and 9% (22/253) among the passive case-finding group (p=0.18). No factors were found to be significantly associated with misclassification. Conclusion: Around one in every ten patients registered as ‘new’ had previous history of TB treatment. Corrective measures need to be implemented, followed by monitoring of any change in the proportion of ‘previously treated’ patients among all registered patients treated under the programme at national level.", "keywords": [ "Tuberculosis/classification", "Previously treated TB", "New TB", "Recurrent TB", "Vulnerable populations" ], "content": "Introduction\n\nIndia has the highest tuberculosis (TB) burden in the world. The annual estimated TB incidence and deaths is 2.7 million and 0.4 million, respectively1. Of the patients receiving treatment under its revised national tuberculosis control programme (RNTCP), the proportion of ‘previously treated’ patients (received anti-TB drugs in the past for one month or more) was 19% in 2016 and 15% in 20172,3. The national anti-tuberculosis drug resistance (2014–16) survey shows that ‘previously treated’ TB patients have four times higher prevalence of multidrug-resistant TB (MDR-TB) when compared to new patients (11.6% versus 2.8%)4.\n\nIn 2007, Atre et al.5 reported 11% misclassification among ‘new’ patients registered under the RNTCP. It is important to know how the programme is faring 10 years down the line. This study was carried out as a part of a larger study among new smear-positive pulmonary TB patients to determine the effectiveness of a community-based active case-finding (ACF) strategy when compared to passive case-finding (PCF) in 18 randomly selected districts of India6,7. The ACF strategy was conducted as part of Project Axshya (meaning ‘free of TB’) whose focus was to increase detection of new smear-positive pulmonary TB patients among marginalised and vulnerable populations. Before enrolling the newly registered TB patients (both ACF and PCF patients) into our study, we enquired about their history of previous treatment. This provided us with a unique opportunity to document the proportion of newly registered smear-positive pulmonary TB patients that had previous history of TB treatment and were therefore misclassified.\n\n\nMethods\n\nThis was a cross-sectional study involving new smear-positive pulmonary TB patients from marginalised and vulnerable populations that were registered for treatment under the RNTCP in India between March 2016 and February 2017.\n\nNational TB programme (2016–17): India’s RNTCP infrastructure included national, state, district and sub-district level administrative units (one for 250 000 to 500 000 population) and designated microscopy centres for sputum smear microscopy8. Before starting TB treatment, the medical officer in the health facility classified the patients as ‘new’ or ‘previously treated’.\n\nDuring the study period (March 2016 to February 2017), new patients received two months of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol followed by four months of Isoniazid, Rifampicin and Ethambutol. ‘Previously treated’ patients received two months of Isoniazid, Rifampicin, Pyrazinamide, Ethambutol and Streptomycin, one month of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol and five months of Isoniazid, Rifampicin and Ethambutol. Among TB patients, a subset of patients who were at high risk to have MDR-TB (presumptive MDR-TB patients) underwent genotypic drug susceptibility testing (DST). These included patients previously treated for TB, patients with a TB-HIV co-infection, patients who upon follow up during TB treatment were smear-positive and contacts of a confirmed MDR-TB patient.\n\nProject Axshya: Project Axshya is implemented in India by the South-East Asia office of the International Union against Tuberculosis and Lung Disease (The Union) to enhance the reach and visibility of RNTCP services among marginalised and vulnerable populations and to mitigate the impact of TB on the country (see Box for criteria for marginalised and vulnerable populations). Axshya SAMVAD (SAMVAD is an acronym for sensitization and advocacy in marginalised and vulnerable areas of the district) is the ACF strategy under the project. The word ‘SAMVAD’ in Sanskrit language means ‘conversation’. In 2016–17, the project covered 285 districts spread across 19 states.\n\n1. Slums\n\n2. Tribal areas\n\n3. Marginalised communities as per the constitution of India\n\n4. In pockets where occupational lung diseases are high\n\n5. In pockets where there is high risk of acquiring TB like; stone crushing/mining/weaving industry/unorganized labour (construction workers etc)/homeless people\n\n6. In pockets reported to have high HIV/ AIDS burden\n\n7. In areas or communities where incidence of TB is high\n\n8. Among household contacts of smear-positive pulmonary TB patients\n\n9. Prisons\n\nTB – tuberculosis; HIV – human immunodeficiency virus; AIDS – acquired immunodeficiency syndrome; Project Axshya –implemented by The Union, South East Asia office, New Delhi, India, across 285 districts of India, to enhance the reach and visibility of national TB programme services among marginalised and vulnerable populations and to mitigate the impact of TB on the country. Axshya in Sanskrit means ‘free of TB’.\n\nAxshya SAMVAD study: This study was conducted among new smear-positive pulmonary TB patients to determine the effectiveness of Axshya SAMVAD on diagnosis and treatment initiation delays, costs due to TB diagnosis and treatment outcomes6,7. We included all new smear-positive pulmonary TB patients from marginalised and vulnerable populations that were detected through ACF and registered under the programme in the 18 randomly sampled Axshya districts (simple random sampling) during March 2016 to February 2017. Every month in the same districts, we randomly sampled an equal number of new smear-positive pulmonary TB patients registered through PCF from marginalised and vulnerable populations (simple random sampling)6,7. Random numbers for simple random sampling were generated using Microsoft Excel.\n\nUnder Axshya SAMVAD study, we collected data for each study participant through record review (age, gender, ACF/PCF status, residence (urban/rural), distance of residence from microscopy centre, sputum smear grade, weight, diabetes status and HIV status) and patient interviews at their residence. Patient interviews were set up during the review of the participant’s record. Before starting the patient interviews, we enquired about their past history of TB treatment for at least one month either from the public or private sector. Those with a past history of treatment were excluded from the Axshya SAMVAD study and referred to the programme for appropriate management. These constitute ‘misclassification’ for the purpose of present analysis.\n\nWe double entered and validated the data using EpiData Entry software9 (version 3.1, EpiData Association, Odense Denmark). We analysed the data using STATA (version 12.1, copyright 1985–2011 StataCorp LP USA)10. We used frequency and proportions (0.95 confidence intervals (CI)) to summarise (infer) the extent of misclassification. Adjusted analysis was done using log binomial regression to determine the factors associated with misclassification. Variables collected during record review (age, gender, ACF/PCF status, residence (urban/rural), distance of residence from microscopy centre and sputum smear grade) were included in the adjusted analysis. Baseline weight was missing in two-fifths of patients; baseline diabetes status was missing for more than three-fifths and only one patient was living with HIV. Hence, we excluded them from the adjusted analysis. The association was summarized (inferred) using adjusted prevalence ratios (0.95 CIs).\n\nThe Axshya SAMVAD study was approved by the Ethics Advisory Group of The Union, Paris, France (EAG number 15/15, dated 28 September 2015). We conducted the study after receiving approvals from the State Tuberculosis Officers in the respective states (18 randomly sampled Axshya districts belonged to seven states). We obtained written informed consent for participation from all the study participants.\n\n\nResults\n\nFigure 1 depicts the misclassification of ‘previously treated’ smear-positive pulmonary TB patients as ‘new’. A total of 629 newly registered smear-positive pulmonary TB patients were enrolled for the Axshya SAMVAD study. We couldn’t contact 108 (17%) for interview as patients were not available at their residence during the visit (a maximum of two visits were made).\n\nTB – tuberculosis; SAMVAD – sensitization and advocacy in marginalised and vulnerable areas of the district; Axshya SAMVAD – an active case- finding strategy under project Axshya, implemented by The Union, South East Asia office, New Delhi, India, across 285 districts of India. registered under programme between March 2016 and February 2017 for treatment after classification as ‘new’.\n\nOf the 521 interviewed, 56 [10.8% (95% CI: 8.4%, 13.7%)] had a past history of TB treatment (public or private) for at least a month: 12.7% (34/268) among the ACF group and 8.7% (22/253) among the PCF group (p=0.18). No factors were found to be significantly associated with misclassification (Table 1).\n\nTB – tuberculosis; SAMVAD – sensitization and advocacy in marginalised and vulnerable areas of the district; Axshya SAMVAD – an active case-finding strategy under project Axshya implemented by The Union, South East Asia office, New Delhi, India, across 285 districts of India; aPR – adjusted prevalence ratio; CI – confidence interval.\n\nregistered under programme between March 2016 and February 2017 for treatment after classification as ‘new’; *Total 661 were enrolled, 32 were later excluded as they did not fit the operational definition of study participant based on information obtained from record review. Among 629 eligible for patient interviews, 521 study participants could be contacted; @log binomial regression.\n\n\nDiscussion\n\nAbout one in ten ‘new’ TB patients had a past history of TB treatment. This misclassification meant that these patients received the wrong treatment regimen as per the national guidelines at the time. This is similar to the RNTCP report of 20183 and previous documentation in 20075. The misclassification among new smear-positive TB patients was two times higher than the 4.5% reported from Malawi in 200011.\n\nOne possible reason for this might be a lack of attention on the part of the medical officer to enquire for previous history of TB before starting treatment. Ambiguity in classification when there was a large gap between previous and current treatment, absence of treatment records and patients’ reluctance to disclose previous treatment details due to possible stigma (fear of being seen as a ‘problem patient’) could be the other reasons5.\n\nThis study has some limitations. First, this programmatically relevant finding was incidental and part of a larger study (Axshya SAMVAD study) and hence, we did not systematically record the details of past TB treatment (when, duration of treatment, whether under programme or in private sector) and the reasons for misclassification. Secondly, as patients with misclassification were excluded from the Axshya SAMVAD study, we do not know what happened to them, including their treatment outcomes. Thirdly, we did not include smear-negative pulmonary TB and extrapulmonary TB patients as they were not part of the Axshya SAMVAD study. In Malawi (2000)11, they had a higher risk of misclassification when compared to smear-positive pulmonary TB patients. Finally, non-response was a limitation. However, in a best-case scenario (assuming all 108 non-responders did not have previous history of TB treatment), the proportion of misclassification would have been 8.9% (56/629) which is still programmatically significant.\n\nLimitations notwithstanding, our study has programme implications. Of the new smear-positive pulmonary TB patients registered in India in 2016, 21% had an unfavourable outcome3. Some of these unfavourable outcomes can be explained by wrong management – patients getting an inferior treatment regimen (previously treated patients being treated with a regimen meant for new cases) and missing an opportunity for drug susceptibility testing (as previously treated patients were eligible for DST at the time).\n\nIndia has recently adopted the World Health Organization (WHO) recommendation that the category II regimen (for ‘previously treated’ patients) ‘should no longer be prescribed and drug susceptibility testing should be conducted to inform the choice of treatment regimen’12. To make this a reality, India now recommends universal DST, meaning all diagnosed TB patients are eligible for testing via the Xpert MTB/RIF assay® (Cepheid Sunnyvale USA) followed by first-line (if rifampicin susceptible) or second-line line probe assay (if rifampicin resistant)4,13. This further means that both new and previously treated patients are treated with the same regimen4,8,14. Hence, in the present scenario, the impact of misclassification on individual patient management is minimal. This was not the case at the time of conduct of this study. Despite these developments, we think asking for previous treatment history is still relevant for two reasons. First, the information on the proportion of previously treated patients is epidemiologically an important piece of information and is regularly reported to the WHO for monitoring the global TB epidemic. Second, the universal DST is not a reality in every part of the country and in such instances, prioritizing previously treated patients for DST is a better strategy, given the higher prevalence of drug-resistant TB among them.\n\nOur findings were based on patients from marginalised and vulnerable populations and this limits our generalisability to TB patients registered from the general population. The programme should consider replicating similar studies among patients from the general population.\n\nSince 2017, the revised laboratory register at the level of designated microscopy centres under the RNTCP (one per 50 000 to 100 000 population) also captures this information of previous treatment8. Hence, complete filling of the revised laboratory register at microscopy centres should be closely monitored by the programme and future operational research should focus on this.\n\nSystematic qualitative enquiry is recommended to understand the ‘why’ (why does it happen) and ‘how’ (how can it be addressed) of misclassification.\n\n\nConclusions\n\nThis study demonstrated that ‘previously treated’ patients were being missed and were being registered as ‘new’ patients under the RNTCP in India. Corrective measures need to be implemented, followed by monitoring any change in the proportion of ‘previously treated’ patients among all registered patients treated under the programme at national level.\n\n\nData availability\n\nfigshare: Underlying data. https://doi.org/10.6084/m9.figshare.775668815\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: Questionnaire Axshya SAMVAD study. https://doi.org/10.6084/m9.figshare.776858916\n\nThis project contains the following extended data:\n\nS2 Annex.pdf (Part I of the questionnaire – record review)\n\nS3 Annex.pdf (Part II of the questionnaire – patient interview)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of the patients’ details was obtained from the patients.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to acknowledge funding support for Project Axshya from The Global Fund TB grant to India. The Project is implemented by the Project Management Unit of The Union South East Asia Office since 2010 till date with support of the sub-recipient partners (in alphabetical order): The Catholic Bishops’ Conference of India-Coalition for AIDS and Related Diseases (CBCI-CARD); The Catholic Health Association of India (CHAI); Emmanuel Hospital Association (EHA); MAMTA Health Institute for Mother and Child; Population Services International (PSI); Resource Group for Education and Advocacy for Community Health (REACH); and Voluntary Health Association of India (VHAI). Publication fee for this study were covered by the Department for International Development (DFID), UK and La Fondation Veuve Emile Metz-Tesch (Luxembourg).\n\nWe thank the following for their support in data collection: Robinson Robert, Madhu Nema, Yashpal Singh Rajput. We would also like to thank other Project Axshya staff: Anand Das, Ganesh M, A Mary Mamatha, Antony Santhappan, Prabhat Kumar Singh, Deepak Tigga and Khumanthem Jayanta Kumar Singh, Kamlesh Kumar and Ranjan Singh who participated in the initial training, planning and/or questionnaire development. We would also like to thank the RNTCP staff in the study districts that supported the District Coordinators and Interpersonal Communication Coordinator in study participant enrolment and record review. We thank the Department for International Development (DFD), UK, for funding the Global Operational Research Fellowship Programme at the International Union Against Tuberculosis and Lung Disease (The Union), Paris, France in which HDS and JPT work as a senior operational research fellow.\n\nDisclaimer: The contents of this paper do not necessarily reflect the views of the Government or Non-Governmental Organizations or The Union\n\n\nReferences\n\nWorld Health Organization (WHO): Global tuberculosis report 2018. Geneva, Switzerland; 2018. Reference Source\n\nRevised National Tuberculosis Control Programme (RNTCP): TB India 2017. Annual status report. New Delhi India; 2017. Reference Source\n\nRevised National Tuberculosis Control Programme (RNTCP): TB India 2018. Annual status report. New Delhi, India; 2018. Reference Source\n\nCentral TB Division: Directorate General of Health Services. Ministry of Health & Family Welfare. Government of India. Guidelines on Programmatic Management of Drug Resistant TB (PMDT) in India. New Delhi, India; 2017. Reference Source\n\nAtre SR, D’Souza DT, Dholakia YN, et al.: Observations on categorisation of new TB cases: implications for controlling drug resistance. Int J Tuberc Lung Dis. 2007; 11(10): 1152–3. PubMed Abstract \| Free Full Text\n\nAxshya SAMVAD Study Group, Shewade HD, Chadha SS, et al.: Data collection using open access technology in multicentre operational research involving patient interviews. Public Health Action. 2017; 7(1): 74–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShewade HD, Gupta V, Satyanarayana S, et al.: Active case finding among marginalised and vulnerable populations reduces catastrophic costs due to tuberculosis diagnosis. Glob Health Action. 2018; 11(1): 1494897. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRevised National Tuberculosis Control Programme (RNTCP); Central TB Division. Ministry of Health and Family Welfare. Government of India. Technical and operational guidelines for tuberculosis control in India. New Delhi India; 2016. Reference Source\n\nLauritsen JM, Bruus M: EpiData Entry (version 3.1). A comprehensive tool for validated entry and documentation of data. The EpiData Association, Odense Denmark, 2004. Reference Source\n\nStataCorp: Stata Statistical Software: Release 12. College Station, TX: StataCorp LP. 2011.\n\nHarries AD, Hargreaves NJ, Kwanjana JH, et al.: Relapse and recurrent tuberculosis in the context of a national tuberculosis control programme. Trans R Soc Trop Med Hyg. 2000; 94(3): 247–9. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization (WHO): Treatment of tuberculosis. Guidelines for treatment of drug susceptible tuberculosis and patient care. 2017 update. Geneva, Switzerland; 2017. Reference Source\n\nCentral TB Division, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India. Guidelines on Programmatic Management of Drug Resistant TB (PMDT) in India. New Delhi, India; 2012. Reference Source\n\nRevised National Tuberculosis Control Programme (RNTCP); Central TB Division: National strategic plan for TB elimination 2017–25. New Delhi, India; 2017. Reference Source\n\nShewade H: Underlying data. figshare; 2019. Reference Source\n\nShewade H: Questionnaire Axshya SAMVAD study. figshare; 2019. Reference Source" }	[ { "id": "46256", "date": "09 Apr 2019", "name": "Sachin Atre", "expertise": [ "Reviewer Expertise Operational research on TB and MDR-TB in India" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addresses one of the crucial problems with in India’s Revised National TB Control Program. Interestingly, the study is based on our prior study (in which I was a lead author) in 2007, which was conducted in Mumbai and rural areas of Pune district. Given the research context and the data, I feel this manuscript should go as a brief communication or notes from the field rather than a full original research article. It does not make any novel contribution, but just confirms the earlier research finding. Even to make it a brief communication, I feel some points need to be provocatively addressed. My comments are as below.\nAuthors have provided details of their larger study, of which the current study is only a part. I feel that level of details is unnecessary here. On the other hand, unfortunately despite having a large team (as seen from the long list of authors), they did not do in-depth inquiry into the reasons for an erroneous categorization of cases, which is actually the main aim of the manuscript. They just mentioned the same reasons for erroneous categorization by providing a reference to our 2007 article without making any new contribution. Identifying the most prominent reasons would have been helpful to identify as a focus area for the policy makers to make some action plan for operational implementation of the program. In my opinion, there is not much substance to publish it as an original research article. Authors nowhere discussed the major implication of their observation that even after 10 years, there remains a big disconnect between the operational research and the program implementation, which is really unfortunate. This finding has another major implication that because of erroneous categorization, previously treated cases are being treated with first-line regimen, which results in amplification of resistance in those cases who may have primary or acquired drug resistance (from prior treatment) and/MDR-TB. This has been happening for over 10 years so one can see why India now faces the serious problem of drug resistant TB. I am not convinced with the factor analysis in Table 1 because the detailed inquiry was not made into reasons for misclassification/erroneous categorization which should have been the main focus. Authors state in discussion that as per the WHO recommendation, universal DST will be done for all TB cases. This is an ideal situation. The scale up of GeneXpert even remains questionable. The WHO global report 2018 showed that only 40% of TB cases in India were subjected to GeneXert in 2017. There are problems of shortage of cartridges, falcon tubes, power outages etc. Given the glacial speed of translation of findings from operational research in the actual RNTCP implementation, India’s claim of TB elimination by 2025 looks really questionable.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4628", "date": "16 May 2019", "name": "Hemant Deepak Shewade", "role": "Author Response", "response": "REVIEWER #1Sachin R. Atre, Johns Hopkins University, USA; Dr. D.Y. Patil Medical College, Hospital and Research Centre Pune, India REVIEWER COMMENTThe manuscript addresses one of the crucial problems with in India’s Revised National TB Control Program. Interestingly, the study is based on our prior study (in which I was a lead author) in 2007, which was conducted in Mumbai and rural areas of Pune district. Given the research context and the data, I feel this manuscript should go as a brief communication or notes from the field rather than a full original research article. It does not make any novel contribution, but just confirms the earlier research finding. Even to make it a brief communication, I feel some points need to be provocatively addressed. My comments are as below.AUTHOR RESPONSEThank you for the comment. F1000Research does not have the article types as suggested: brief communication or notes from the field. ‘Research notes’ is the closest option, but here too there is no specific word count mentioned. Considering our manuscript is around 1800 words only, we are fine if F1000Research agrees to consider this as a research note.We agree the findings are not novel, but are timely considering 10 years down the line, the issue remains as it is and this is important to highlight.We have addressed your comments, details below. REVIEWER COMMENTAuthors have provided details of their larger study, of which the current study is only a part. I feel that level of details is unnecessary here. On the other hand, unfortunately despite having a large team (as seen from the long list of authors), they did not do in-depth inquiry into the reasons for an erroneous categorization of cases, which is actually the main aim of the manuscript. They just mentioned the same reasons for erroneous categorization by providing a reference to our 2007 article without making any new contribution. Identifying the most prominent reasons would have been helpful to identify as a focus area for the policy makers to make some action plan for operational implementation of the program. In my opinion, there is not much substance to publish it as an original research article.AUTHOR RESPONSEThe actual study was done across 18 randomly spread districts of the country (Axshya SAMVAD study – Axshya SAMVAD is an ACF strategy for detecting TB and is implemented by The Union South East Asia). As mentioned, the findings were accidental (not intended) and we thought it is important to report this and look for differences in rates of misclassification across various patient sub-groups.The Axshya SAMVAD study was conducted by project Axshya staff in operational setting without any additional funding for the study itself. The research team in the field (project Axshya staff) were not trained and did not have the required capacity to conduct qualitative research. Hence, we could not go in-depth into the reasons. But, one key point is that our study was conducted in randomly selected 18 districts of the country and hence, the findings are representative.We agree the findings are not novel, but are timely considering 10 years down the line, the issue remains as it is and this is important to highlight.Considering our manuscript is around 1800 words only, we are fine if F1000Research agrees to consider this as a research note. REVIEWER COMMENTAuthors nowhere discussed the major implication of their observation that even after 10 years, there remains a big disconnect between the operational research and the program implementation, which is really unfortunate. This finding has another major implication that because of erroneous categorization, previously treated cases are being treated with first-line regimen, which results in amplification of resistance in those cases who may have primary or acquired drug resistance (from prior treatment) and/MDR-TB. This has been happening for over 10 years so one can see why India now faces the serious problem of drug resistant TB.AUTHOR RESPONSEThank you very much for your comment. We agree with the reviewer. We have included the above point in the revised manuscript as suggested by the reviewer. We have discussed the implications in the first paragraph of ‘Implications for the TB programme’ in the revised manuscript (reproduced below):“Limitations notwithstanding, our study has programme implications. Of the new smear-positive pulmonary TB patients registered in India in 2016, 21% had an unfavourable outcome 3 . Some of these unfavourable outcomes can be explained by wrong management – patients getting an inferior treatment regimen (previously treated patients being treated with a regimen meant for new cases) and missing an opportunity for drug susceptibility testing (as previously treated patients were eligible for DST at the time). Inferior regimen might have also contributed to amplification of resistance in those who may have primary or acquired drug resistance (from prior treatment) and MDR-TB. This has been happening for over 10 years so one can see why India now faces the serious problem of drug resistant TB.”REVIEWER COMMENTI am not convinced with the factor analysis in Table 1 because the detailed inquiry was not made into reasons for misclassification/erroneous categorization which should have been the main focus.AUTHOR RESPONSEThank you for the comment. In Table 1, we are quantitatively looking for patient sub-groups who have higher prevalence of misclassification. Though not statistically significant, programmatically significant differences were observed in misclassification in urban and rural areas (12% in rural and 2% in urban areas). We agree that we have not looked into the ‘why’ and ‘how’ of misclassification (limitations). But, based on our programme experiences made some useful recommendations in the revised manuscript (reproduced below)“Our findings were based on patients from marginalised and vulnerable populations and this limits our generalisability to TB patients registered from the general population. The programme should consider replicating similar studies among patients from the general population with a possible sub-group to look for rural-urban differences.Since 2017, the revised laboratory register at the level of designated microscopy centres under the RNTCP (one per 50 000 to 100 000 population) also captures this information of previous treatment 8 . RNTCP staff needs to be re-sensitized to “ask” for previous history of TB treatment. Hence, complete filling of the revised laboratory register at microscopy centres should be closely monitored by the programme and future operational research should focus on this.Systematic qualitative enquiry is recommended to understand the ‘why’ (why does it happen) and ‘how’ (how can it be addressed) of misclassification. In the national case-based TB notification software (NIKSHAY), record linkage and deduplication using key attributes may be considered to identify repeat notification of the same person separated by a time period.” REVIEWER COMMENTAuthors state in discussion that as per the WHO recommendation, universal DST will be done for all TB cases. This is an ideal situation. The scale up of GeneXpert even remains questionable. The WHO global report 2018 showed that only 40% of TB cases in India were subjected to GeneXert in 2017. There are problems of shortage of cartridges, falcon tubes, power outages etc. Given the glacial speed of translation of findings from operational research in the actual RNTCP implementation, India’s claim of TB elimination by 2025 looks really questionable.AUTHOR RESPONSEThank you for the comment. We agree with the reviewer. We agree that DST for all TB patients is an ideal situation. Therefore, we have also stated that in a non-ideal situation (DST not possible for all TB patients), DST may be focussed on those with previous history of TB treatment. Therefore, correct classification as new or previously treated is still relevant. We have mentioned this in the last three lines of the section ‘implications for the TB programme’ (reproduced below):“Second, the universal DST is not a reality in every part of the country and in such instances, prioritizing previously treated patients for DST is a better strategy, given the higher prevalence of drug-resistant TB among them.”" } ] }, { "id": "46257", "date": "12 Apr 2019", "name": "Otavio T. Ranzani", "expertise": [ "Reviewer Expertise Epidemiology", "Infection", "Tuberculosis", "Pneumonia", "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\nThe authors conducted a secondary analysis on the data originated from the Axshya SAMVAD study aiming to describe the proportion of patients with smear-positive pulmonary tuberculosis labelled as \"new\" (regarding antibiotic treatment), but that actually have had tuberculosis before. The research question is of public health importance, it is not widely reported in the literature, and makes this short article interesting to the literature. The authors should be commended. I have some comments below aiming to improve the manuscript.\nThe authors should clarify to the reader on abstract that \"new\" refers to previous TB treatment for at least 30 consecutive days. The authors should revise the entire manuscript, particularly Abstract and Introduction, to clarify about what \"missclassification\" refers to. Misclassification is an epidemiological term, but it can refer to different variables depending on the context. And during the read, it became clear only after going to the results section. Example: (abstract) \"reported 11% misclassification among ‘new’ patients\". Maybe the authors could rephrase as:\"reported that 11% of patients with tuberculosis were misclassified as new patients regarding previous treatment history\" or \"reported that 11% of patients with tuberculosis were misclassified as new patients despite their previous TB treatment\" or \"reported that 11% of patients with tuberculosis were misclassified as new patients despite previously treated\" Regarding the inclusion criteria, did it only include adults? Regarding Table 1: Why did you categorize age? And why in only 3 categories merging different age profile? For instance, the range 15-44 covers different population. You might have loose power and contrast. You should use age as continuous and/or open the category 15-44. Do the authors have converge problems with the log-binomial model? The authors should re-phrase their statements on the prevalence ratio associated factors on Table 1. For instance, it is clear that Residence (rural/urban) is an important factor, with a big point estimate, but wide confidence interval (6.4 (0.9, 48.2)). Likely with a bigger sample size and/or higher number of events, you would have better precision and would achieve \"significancy\" or \"no include 1\". How is it possible to have such low prevalence of positive HIV status in a vulnerable population? How was the HIV testing coverage? The discussion section must be improved: Although recommended and ideal, DST will not be available for all. Even if available, the previous history treatment is fundamental. The authors should propose solutions for the problem, such as linkage deduplication, linked records form the national system, etc. Please, better discuss the potential selection bias and representative of the included population regarding India population.\nMinor:\nMaybe better to use the notation of 95% CI rather than 0.95 Figure 1. I think the first box at left has a wrong \"the\" : as AN error Change gender to sex\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4629", "date": "16 May 2019", "name": "Hemant Deepak Shewade", "role": "Author Response", "response": "REVIEWER #2Otavio T. Ranzani, University of São Paulo, Brazil; Barcelona Institute for Global Health (ISGlobal), Spain REVIEWER COMMENTThe authors conducted a secondary analysis on the data originated from the Axshya SAMVAD study aiming to describe the proportion of patients with smear-positive pulmonary tuberculosis labelled as \"new\" (regarding antibiotic treatment), but that actually have had tuberculosis before. The research question is of public health importance, it is not widely reported in the literature, and makes this short article interesting to the literature. The authors should be commended. I have some comments below aiming to improve the manuscript.AUTHOR RESPONSEThank you very much for the constructive comments. REVIEWER COMMENTThe authors should clarify to the reader on abstract that \"new\" refers to previous TB treatment for at least 30 consecutive days.AUTHOR RESPONSEWe are not clear here what the reviewer intends to mean. ‘New’ refers patients who have not been previously treated for TB (at least for one month). REVIEWER COMMENTThe authors should revise the entire manuscript, particularly Abstract and Introduction, to clarify about what \"misclassification\" refers to. Misclassification is an epidemiological term, but it can refer to different variables depending on the context. And during the read, it became clear only after going to the results section. Example: (abstract) \"reported 11% misclassification among ‘new’ patients\". Maybe the authors could rephrase as: \"reported that 11% of patients with tuberculosis were misclassified as new patients regarding previous treatment history\" or \"reported that 11% of patients with tuberculosis were misclassified as new patients despite their previous TB treatment\" or \"reported that 11% of patients with tuberculosis were misclassified as new patients despite previously treated\"AUTHOR RESPONSEThank you for the comment. In the revised manuscript, both at beginning of abstract and main text introduction, we have clarified as to what we mean by ‘misclassification’ (reproduced below)Abstract“In 2007, a field observation from India reported 11% misclassification among ‘new’ patients registered under the revised national tuberculosis (TB) control programme. Ten years down the line, it is important to know what proportion of newly registered patients has a past history of TB treatment for at least one month (henceforth called as ‘misclassification’).” Introduction (main text)“This provided us with a unique opportunity to document the proportion of newly registered smear-positive pulmonary TB patients that had previous history of TB treatment and were therefore misclassified (henceforth called as ‘misclassification’).” REVIEWER COMMENTRegarding the inclusion criteria, did it only include adults?AUTHOR RESPONSEThank you for pointing this out. Yes it included only adults (≥ 15 y). We have clarified this under study population and titles of tables and figures. REVIEWER COMMENTRegarding Table 1: Why did you categorize age? And why in only 3 categories merging different age profile? For instance, the range 15-44 covers different population. You might have loose power and contrast. You should use age as continuous and/or open the category 15-44. Do the authors have converge problems with the log-binomial model? The authors should re-phrase their statements on the prevalence ratio associated factors on Table 1. For instance, it is clear that Residence (rural/urban) is an important factor, with a big point estimate, but wide confidence interval (6.4 (0.9, 48.2)). Likely with a bigger sample size and/or higher number of events, you would have better precision and would achieve \"significancy\" or \"no include 1\".AUTHOR RESPONSEThank you for the comment. We categorized aged, because we wanted to explore whether misclassification was significantly higher among certain meaningful sub-groups. Regarding opening up the category 15-44 y, we decided not to do this as there are only 25 events of interest (=misclassification) in this sub-group. If we further divide this sub-group, the event of interest could get reduced and this would have further widened the 95% CI for the adjusted prevalence ratios.As per reviewer comments, we have added a line under results narrative in the revised manuscript to discuss at adjusted prevalence ratio for the variable ‘residence’ (reproduced below).“Patients belonging to rural areas had higher prevalence of misclassification when compared to urban areas (12% vs 2%), but this difference was not statistically significant probably due to small sample size.” REVIEWER COMMENTHow is it possible to have such low prevalence of positive HIV status in a vulnerable population? How was the HIV testing coverage?AUTHOR RESPONSEThe HIV percentage among all TB patients in India is around 3%. In our study, HIV status was missing in records for two-fifth patients and among those whose test results were recorded (n=288), one was positive. REVIEWER COMMENTThe discussion section must be improved: Although recommended and ideal, DST will not be available for all. Even if available, the previous history treatment is fundamental. The authors should propose solutions for the problem, such as linkage deduplication, linked records form the national system, etc. Please, better discuss the potential selection bias and representative of the included population regarding India population.AUTHOR RESPONSEThank you very much. We have reviewed and revised the discussion section as per the comments.We agree that DST may not be available for all. Hence, in the “Implications for the TB programme” sub-section under discussion section we have included this this. We are reproducing it below:“Second, the universal DST is not a reality in every part of the country and in such instances, prioritizing previously treated patients for DST is a better strategy, given the higher prevalence of drug-resistant TB among them.” Regarding DST availability, we are reproducing the relevant discussion lines below“Hence, in the present scenario, the impact of misclassification on individual patient management is minimal. This was not the case at the time of conduct of this study. Despite these developments, we think asking for previous treatment history is still relevant for two reasons. First, the information on the proportion of previously treated patients is epidemiologically an important piece of information and is regularly reported to the WHO for monitoring the global TB epidemic. Second, the universal DST is not a reality in every part of the country and in such instances, prioritizing previously treated patients for DST is a better strategy, given the higher prevalence of drug-resistant TB among them” (second paragraph under ‘implications for TB programme’ sub-section of discussion)Regarding potential selection bias and representativeness, we are reproducing the relevant discussion lines below:“Our findings were based on patients from marginalised and vulnerable populations and this limits our generalisability to TB patients registered from the general population. The programme should consider replicating similar studies among patients from the general population with a possible sub-group to look for rural-urban differences.” (First paragraph under ‘recommendations’ sub-section of discussion)Regarding proposing solutions, we have revised it as per reviewer suggestions under recommendations subsection of discussion (reproducing below)“Since 2017, the revised laboratory register at the level of designated microscopy centres under the RNTCP (one per 50 000 to 100 000 population) also captures this information of previous treatment 8 . RNTCP staff needs to be re-sensitized to “ask” for previous history of TB treatment. Hence, complete filling of the revised laboratory register at microscopy centres should be closely monitored by the programme and future operational research should focus on this.Systematic qualitative enquiry is recommended to understand the ‘why’ (why does it happen) and ‘how’ (how can it be addressed) of misclassification. In the national case-based TB notification software (NIKSHAY), record linkage and deduplication using key attributes may be considered to identify repeat notification of the same person separated by a time period.”REVIEWER COMMENTMinor:Maybe better to use the notation of 95% CI rather than 0.95AUTHOR RESPONSEWe have revised throughout the manuscript as per reviewer suggestion REVIEWER COMMENTFigure 1. I think the first box at left has a wrong \"the\" : as AN errorAUTHOR RESPONSEWe are not clear as to what the reviewer means here. We did not find this error in the first left box of figure 1. However, we re-checked figure 1 and found it no grammatical errors. REVIEWER COMMENTChange gender to sexAUTHOR RESPONSEWe have revised throughout the manuscript as per reviewer suggestion" } ] } ]	1	https://f1000research.com/articles/8-338
https://f1000research.com/articles/8-669/v1	16 May 19	{ "type": "Research Article", "title": "Thyroid hormonal changes among women with polycystic ovarian syndrome in Baghdad – a case-control study", "authors": [ "Mayada M. Moustafa", "Mohammed Y. Jamal", "Rawaa Dawood Al-Janabi", "Mayada M. Moustafa", "Rawaa Dawood Al-Janabi" ], "abstract": "Background: Polycystic ovarian syndrome is a syndrome of ovarian dysfunction along with the cardinal features of hyperandrogenism and polycystic ovary morphology. The prevalence of polycystic ovaries on ultrasound is around quarter of all women but is not associated with the full syndrome. The study aimed to assess the status of thyroid disorders in polycystic ovarian syndrome (PCOS). Methods: This prospective hospital-based case-control study involved most outpatients aged 13–45 years who visited the Obstetrics, Gynecology, and Infertility clinic at Baghdad Teaching Hospital with complaints of hirsutism and/or oligomenorrhea or infertility. This study included 70 patients, including 50 with PCOS (PCOS group) and 20 without (control group). Results: The PCOS group exhibited significantly higher mean thyroid stimulating hormone level (3.9 vs. 3.1 µIU/L), luteinizing hormone level (15.2 vs. 4.7 mIU/mL), and body mass index (28.6 vs. 24.9 kg/m2; all, p<0.001) and a non-significantly higher follicle-stimulating hormone level (9.2 vs. 5.2 mIU/L) than the control group. Conclusion: Our results demonstrate a higher prevalence of thyroid disorder among women with PCOS.", "keywords": [ "Polycystic ovary syndrome", "subclinical hypothyroidism", "thyroid hormone" ], "content": "Introduction\n\nPolycystic ovarian syndrome (PCOS) is a condition of ovarian dysfunction characterized by hyperandrogenism and polycystic ovaries. The global prevalence of polycystic ovaries among women is 25%1. PCOS is a state of insulin resistance, which is considered to be the main factor contributing to development of the syndrome; diagnosis is based on the presence of two out of three of the following: clinical and/or biochemical androgen excess, anovulation and polycystic ovaries on pelvic ultrasound2. The mechanisms behind these include hyperinsulinemia, disruption of the hypothalamic–pituitary–gonadal axis, dysregulation of ovarian steroidogenesis, as well as genetic and environmental factors2,3. PCOS mainly affects women in aged 18–353,4. Previous studies have documented ovarian enlargement and cystic transformation in thyroid disorders5–8. Thyroid disorders and PCOS are of widespread in the general population, however, the precise nature of the relationship between the two disorders is not currently know. Although the pathophysiology of thyroid disorder and PCOS are totally different6. Whether this is due to some common factors predisposing an individual to both disorders, or due to a pathophysiological connection between the two disorders has yet to be established5. Two factors making the picture more interesting, are that both have different etiopathology, and that reportedly thyroid disorders are more common in PCOS subjects7–9. PCOS begins soon after menarche age as a endocrinologic abnormality, two most common endocrine symptoms are chronic elevation of luteinizing hormone (LH) and insulin resistance4,7,9. The genetic cause of high LH is unknown. Interestingly, neither an elevation in LH nor insulin resistance alone is enough to initiate the PCOS7,9. High LH and hyperinsulinemia work synergistically, causing ovarian growth, androgen production, and ovarian cyst formation4,5,9. The thyroid gland regulates the rate at which the body converts food for energy, functioning as a thermostat to control the body’s metabolism and other systems. If it secretes hormones too fast will increase metabolism and lead to hyperthyroidism, the inverse leads to slow metabolism, resulting in weight gain and hypothyroidism4,5. However, it is not yet known whether this is because of factors predisposing an individual to both disorders or a pathophysiological connection between the two disorders.\n\n\nMethods\n\nThis hospital-based case-control study was conducted at the Obstetrics, Gynecology, and Infertility clinic at Baghdad Teaching Hospital. The study took place from January to October 2018. We obtained a medical and surgical history, a complete menstrual history, including menarche and family history of PCOS, and history of hirsutism, acne, alopecia, menstrual irregularities, or infertility, also history about last pregnancy and abortion. Any history of headaches or blurred vision, any signs or symptoms of thyroid dysfunction include acne, hirsutism, deepening of the voice, and increase in muscle mass were recorded. Thyroid hormone levels were tested to rule out thyroid disease as an etiology of anovulation, and LH and follicle-stimulating hormone (FSH) also analyzed.\n\nThe study included 50 subjects diagnosed with PCOS and 20 control subjects, who consented to participate of individuals attending the hospital for follow up, treatment and further evaluation. In accordance with the Rotterdam criteria10, PCOS was defined by the presence of any two of the following conditions\n\nParticipant inclusion criteria:\n\n1. Irregular menstruation: no menses in the past 6 months or menstrual cycle prolonged for more than 35 days\n\n2. Increased androgen levels and/or acne and/or alopecia (androgenic pattern)11 or biochemical hyperandrogenism (testosterone level >2.0 nmol/L)\n\n3. Polycystic ovaries (follicles 2–9 mm in diameter and ≥12 in number or ovarian volume ≥10 cm3) identified by transabdominal pelvic ultrasonography after excluding other diseases such as congenital adrenal hyperplasia and virilizing tumors12.\n\nParticipant exclusion criteria:\n\n1. Patients use steroids.\n\n2. Patients on contraceptive pills.\n\n3. Pregnancy\n\n4. Very low body mass index by measuring BMI [Normal (18.6–24.9)m2/Kg, and below that is underweight].\n\n5. Hyperthyroidism, or hypothyroidism (TSH ; normal (0.35 to 5 mU/L), T4; normal (6–12 μg/d), T3; normal (260–480 pg/mL) tests).\n\n6. Neoplasia: thyroid or adrenal (cancer diagnosis via lab tests as above and imaging as MRI, CT scan, PET scan and thyroid scan).\n\nControl inclusion criteria:\n\n1. Healthy. Good physical, mental, or emotional state\n\n2. Not using of any form of medication.\n\n3. Good performance status.\n\nControl exclusion criteria:\n\n1. Diabetic.\n\n2. Positive past-medical history (hypertension, cardiovascular diseases, renal diseases, etc.).\n\n3. Positive past-surgical history (any surgical procedures as thyroidectomy, nephrectomy, hysterectomy etc.). The control haven’t any diseases or operations in past history.\n\nAll patients were assessed by complete history-taking and clinical examination include general, inspection, palpation, auscultation, neurologic, and ophthalmologic examination. For thyroid hormone analysis, 5 mL of venous blood was collected from each patient, at the Obstetrics, Gynecology, and Infertility clinic at Baghdad Teaching Hospital, and when patients attended hospital. This was performed by lab staff by using tourniquets and syringe to collect venous blood. All samples collected from venous blood from arm into tubes. Samples were checked for complete clot formation prior to centrifugation, and for particulate matter prior to analysis. If the assay was performed within 24 hours after collection, the specimen was stored at 2–8°C. If testing was delayed more than 24 hours, the specimen was separated from the clot or red blood cells and stored frozen (–10°C or colder). Specimens were mixed thoroughly after thawing, by gently inverting, and then centrifuged, to ensure consistency in the results. Special care must be taken to prevent contamination. 150 µl of specimen was the minimum volume required to perform the assay. The dilution was performed so that the diluted test results read greater than the sensitivity of the assay, and the concentration of hormones were determined by multiplying the concentration of the diluted sample by the dilution factor (conc. x 10 times dilution). Hormone analysis included estimation of serum free triiodothyronine (T3) [LOT No.: 004206], free tetraiodothyronine (T4) [LOT No.: 003192], thyroid stimulating hormone (TSH) [LOT No.: 001285], luteinizing hormone (LH) [LOT No.: 004211], follicle-stimulating hormone (FSH) [LOT No.: 003701], progesterone, and estradiol [LOT No.: 005481], all these tests measured after collect blood from patients and controls, using (SIEMENS/ ADVIA Centaur®) REF: 03852677 (112219) SMN by Siemens healthcare diagnostics Ltd.\n\nData entry and analysis were performed by using SPSS version 23. Numerical data were expressed as mean±standard deviation and categorical data as percentage. The level of significance, set at p≤0.05, was confirmed by the Student t-test.\n\nThe Medical Ethical Committee of Baghdad University / College of Pharmacy approved this study (code:100123). Written informed consent was taken from participants upon presentation to the hospital to both participate in the study and for the research team to access their medical records.\n\n\nResults\n\nThe average age of participants was 27.7±4.7 years for the PCOS group and for the control group 26.8±4.7 years. All patients lived in urban cities in Baghdad province. The PCOS group exhibited significantly higher mean body mass index (BMI; 28.6 vs. 24.9 kg/m2) and LH level (15.2 vs. 4.7 mIU/mL) and a non-significantly higher FSH level (9.2 vs. 5.2 mIU/L) than the control group, (P-values <0.001, <0.001, <0.007, <0.001, respectively) (Table 1 and Underlying data13). There was a significant association between (P-value <0.003) increased body weight and PCOS; while 86% of patients in the PCOS group were overweight or obese, the proportion of overweight/obese patients in the control group did not exceed 50% (Table 2, Table 3). The proportion of patients with elevated TSH levels was significantly greater in the PCOS group than in the control group (52% vs. 10%). At the same time, it is was significant to find that one-fourth of patients in the PCOS group (24%) showed decreased T3 levels (compared to 0% in the control group). There was a significant and direct correlation between age and T4 level, with increase in age being associated with a coefficient of increase of 0.238 in T4 level. This association was significant only in the PCOS group, in which increase in age was associated with a coefficient of increase of 0.294 per year in T4 level (P-value <0.001), (Table 3). Thyroid function parameters (TSH, T3, and T4 levels) were not correlated with BMI or LH or FSH level. Among the 50 patients with PCOS, 20 (40.4%) had subclinical hypothyroidism (SCH) and 30 (59.6%) were euthyroid (P-values <0.003, <0.001), (Table 4).\n\nPCOS, polycystic ovarian syndrome; LH, luteinizing hormone; FSH, follicle-stimulating hormone; TSH, thyroid-stimulating hormone; BMI, body mass index; T3, triiodothyronine hormone; T4, thyroxine hormone.\n\nE2, Estradiol; AMH, Anti-Mullerian Hormone.\n\nPCO, polycystic ovaries, TSH, thyroid-stimulating hormone; T3, triiodothyronine hormone; and T4, thyroxine hormon.\n\nPCOS, poly cystic ovary syndrome; TSH, thyroid-stimulating hormone; T3, triiodothyronine hormone; and T4, thyroxine hormone.\n\n\nDiscussion\n\nPCOS is the most common disorder among young women and an important cause of infertility in this age group. PCOS and thyroid disorder are two of the most common endocrine disorders in women, and while these conditions are very different. Hypothyroidism, is more common in women with PCOS than in the general population. Thyroid and PCOS are interconnected by both genetic and environmental factors which are believed to be contributing to thyroid disorders in PCOS, and is known to cause PCOS-like ovaries and overall worsening of PCOS and insulin resistance.\n\nThe most obvious connection between thyroid diseases and PCOS seem to be an increase in BMI, which is very prevalent in women with PCOS, observed than control group, (BMI for PCOS =28.6±4.0; BMI for control= 24.9±3.0 with P-value=<0.001).\n\nIn the present study, 20 (40%) of 50 patients with PCOS showed SCH. In a previous study, Michalakis et al. reported an SCH prevalence of 23% among patients seeking treatment for infertility, while another study reported a prevalence of 17.5% among patients with PCOS14.\n\nA few studies have previously analyzed the prevalence of SCH in subjects with PCOS. Subclinical hypothyroidism is observed among women with PCOS, with an estimated prevalence range of 10–25%9. Regarding the impact of subclinical hypothyroidism on the clinical, hormonal or metabolism of women with PCOS, a recent meta-analysis has shown that the coexistence of SCH and PCOS leads to mild alterations in serum lipids, but not in hormone levels (TSH, FSH, LH and their ratio)9.\n\nThe findings of the present study are similar to those of a study by Enzevaei et al. in Iran, where 25.5% of subjects with PCOS were found to have SCH15. Similarly, in a study by Sinha et al. in India, 22.5% subjects with PCOS were reported to have SCH compared to 8.75% in controls and thyroid antibodies have been shown to be present in 27% of patients with PCOS versus 8% in controls16, also indicated the presence of elevated—T3, T4, TSH in patients with PCOS17. Kachuei et al. have also reported a significantly higher prevalence of anti-thyroglobulin antibodies in subjects with PCOS than in control subjects in an Iranian population18.\n\nExamination and radiology investigations alone is not a reliable test to determine PCOS. TSH measures, T4, and T3 may be more applicable in the diagnosis of PCOS. Relying the combinations of all these is sufficient to make an accurate diagnosis and reason why so many people with PCOS and hypothyroid are not misdiagnosed.\n\n\nConclusion\n\nWe conclude that most patient with PCOS will have some degree of thyroid dysfunction, especially SCH. PCOS is much more than just oligomenorrhea, amenorrhea, or infertility. Doctors must be aware of the risk factors for PCOS and intervene with a preventive approach, which may restore normal menstrual function, ovulation, and fertility. Therefore, physicians should consider screening for thyroid function tests at PCOS diagnosis, even in the absence of symptoms related with thyroid dysfunction.\n\n\nData availability\n\nZenodo: Thyroid hormonal changes among women with polycystic ovarian syndrome in Baghdad. http://doi.org/10.5281/zenodo.258928213\n\nThis project contains the following underlying data:\n\nThyroid and PCOS.xlsx (raw thyroid hormone levels for cases and controls)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHelen B: Hormonal control in mensturual cycle and hormonal disorder. In: Helen B and Louise K (ed.) Gynecology. 20ed. CRC Press Taylor & Francis Group; 2017.\n\nLamberg BA: Glucose metabolism in thyroid disease. Acta Med Scand. 1965; 178(3): 351–62. PubMed Abstract\n\nAzziz R, Carmina E, Dewailly D, et al.: Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab. 2006; 91(11): 4237–45. PubMed Abstract \| Publisher Full Text\n\nAdams J, Polson DW, Franks S: Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br Med J (Clin Res Ed). 1986; 293(6543): 355–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinha U, Sinharay K, Saha S, et al.: Thyroid disorders in polycystic ovarian syndrome subjects: A tertiary hospital based cross-sectional study from Eastern India. Indian J Endocrinol Metab. 2013; 17(2): 304–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenetti-Pinto CL, Berini Piccolo VR, Garmes HM, et al.: Subclinical hypothyroidism in young women with polycystic ovary syndrome: an analysis of clinical, hormonal, and metabolic parameters. Fertil Steril. 2013; 99(2): 588–92. PubMed Abstract \| Publisher Full Text\n\nRamanand SJ, Ghongane BB, Ramanand JB, et al.: Clinical characteristics of polycystic ovary syndrome in Indian women. Indian J Endocrinol Metab. 2013; 17(1): 138–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanssen OE, Mehlmauer N, Hahn S, et al.: High prevalence of autoimmune thyroiditis in patients with polycystic ovary syndrome. Eur J Endocrinol. 2004; 150(3): 363–9. PubMed Abstract \| Publisher Full Text\n\nBarberi RL: Induction of ovulation in infertile women with hyperandrogenism and insulin resistance. Am J Obstet Gynecol. 2000; 183(6): 1412–1418. PubMed Abstract \| Publisher Full Text\n\nRotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group: Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004; 19(1): 41–47. PubMed Abstract \| Publisher Full Text\n\nCarmina E, Lobo RA: Treatment of hyperandrogenic alopecia in women. Fertil Steril. 2003; 79(1): 91–95. PubMed Abstract \| Publisher Full Text\n\nDewailly D, Hieronimus S, Mirakian P, et al.: Polycystic ovary syndrome (PCOS). Ann Endocrinol (Paris). 2010; 71(1): 8–13. PubMed Abstract \| Publisher Full Text\n\nJamal MY: Thyroid hormonal changes among women with polycystic ovarian syndrome in Baghdad. 2019. http://www.doi.org/10.5281/zenodo.2589282\n\nMichalakis KG, Mesen TB, Brayboy LM, et al.: Subclinical elevations of thyroid-stimulating hormone and assisted reproductive technology outcomes. Fertil Steril. 2011; 95(8): 2634–2637. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnzevaei A, Salehpour S, Tohidi M, et al.: Subclinical hypothyroidism and insulin resistance in polycystic ovary syndrome: is there a relationship? Iran J Reprod Med. 2014; 12(7): 481–46. PubMed Abstract \| Free Full Text\n\nSinha U, Sinharay K, Saha S, et al.: Thyroid disorders in polycystic ovarian syndrome subjects: A tertiary hospital based cross-sectional study from Eastern India. Indian J Endocrinol Metab. 2013; 17(2): 304–309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTudose TI, Zelenetskaia VS, Kozlov GI, et al.: [Lactotropic and thyrotropic functions of the hypophysis in polycystic ovary syndrome]. Probl Endokrinol (Mosk). 1986; 32(3): 3–7. PubMed Abstract\n\nKachuei M, Jafari F, Kachuei A, et al.: Prevalence of autoimmune thyroiditis in patients with polycystic ovary syndrome. Arch Gynecol Obstet. 2012; 285(3): 853–856. PubMed Abstract \| Publisher Full Text" }	[ { "id": "57228", "date": "10 Dec 2019", "name": "Maria Laura Monzani", "expertise": [ "Reviewer Expertise Thyroid", "PCOS" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper Mayada et al. analyzed the prevalence of thyroid disorders in PCOS patients and controls prospectively enrolled at the Baghdad Teaching Hospital. The study included 70 women aged between 13 and 45 years old (50 PCOS patients and 20 controls) and for each of them an accurate personal and familiar history, details about menstrual cycle, symptoms of hyperandrogenemia and thyroid disorders were collected. Moreover, BMI was calculated and LH, FSH, TSH, T3 and T4 were measured. The study has several points to be improved or better elucidated.\nMajor comments:\nThe study is methodologically limited by the small number of enrolled patients, unclear and different inclusion/exclusion criteria for cases and controls, the total absence of normal values and inaccurate citation of literature on this item.\n\nIn the introduction the pathophysiology of PCOS should be described in a clearer way, distinguishing between risk factors of PCOS and consequences of hyperinsulinemia. Both in abstract and introduction, PCOS diagnosis criteria must be clearly defined, according to Rotterdam criteria.\n\nStudy design and setting: lines 9-10. The listed symptoms are not symptoms of thyroid dysfunction. Are the authors endogrinologists? I suggest to reconsider the whole study with colleagues more expert in gland function and alteration.\n\nStudy design and setting, line 7: \"last pregnancy\". It is better to consider previous pregnancies and abortions.\n\nIn order to have information about axis functionality, estrogen and progesterone should be measured too. Moreover, the authors should specify in which phase of the menstrual cycle (when cycle is present) blood test was performed. They have to give more methodological details: when the blood test was performed (morning? fasting?). Which methods were used to measure hormones in the lab? Normal ranges?\n\nAlso FSH is significantly different among cases and controls.\n\nTable 1: please specify how values are expressed, mean? min-max? median? When min-max are reported, also median should be given. Inidcate normal values.\n\nThe authors state that TSH \"was significantly greater\" in PCOS subjects. What do they mean? Higher than what? Than controls? Than normal ranges?\n\nIn table 3, there should be clearly defined criteria for “elevated” and “decreased”. These are not scientific terms\n\nAt the end of results, the authors wrongly refer to Table 4.\n\nReference 9 is inappropriate.\n\nEnd of discussion: do the authors mean that thyroid hormones are useful in PCOS diagnosis? I completely disagree.\n\nConclusions: It seems that if subclinical hypothyroidism is diagnosed and treated, PCOS is cured. It is a wrong message and not supported by data from the present study\n\nMinor comments:\nReference 2 is not correct. Please provide reference of the first published Rotterdam criteria.\n\nIntroduction, line 9: \"disruption\" is not acceptable. Alterate function is better.\n\n“The genetic cause of high LH is unknown”. In the AACE guidelines a possible explanation is suggested.\n\nEnd of introduction: \"If it secretes hormones too fast\". Too fast is not a scientific term, please rephrase.\n\nFree thyroid hormones where measured, thus the acronyms fT3 and fT4 should be used\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "64723", "date": "24 Jun 2020", "name": "Ibrahim A. Abdelazim", "expertise": [ "Reviewer Expertise Obstetrics", "Gynecology and Reproductive 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\nThank you to give me the opportunity to review, and comment on the article entitled (Thyroid hormonal changes among women with polycystic ovarian syndrome in Baghdad - a case-control study) published by Moustafa et al.\nThe article discussing an interesting subject, and aimed to assess the status of thyroid disorders in polycystic ovarian syndrome (PCOS).\nMoustafa et al., stated in the introduction section that the global prevalence of polycystic ovaries among women is 25%1. While, others mentioned that the PCOS affects 15-20% of women when the ESHRE/ASRM diagnostic criteria used2.\n\nMoustafa et al., mentioned that PCOS is a state of insulin resistance, which is considered to be the main factor contributing to development of the syndrome; diagnosis is based on the presence of two out of three of the following: clinical and/or biochemical androgen excess, anovulation and polycystic ovaries on pelvic ultrasound3.\nWhile, other authors mentioned that PCOS has a reproductive manifestations (anovulation, and hyperandrogenism), and adverse metabolic outcome (insulin resistance (IR), and glucose intolerance)4'5. So, IR is not a constant finding of PCOS, and it is only a manifestation of the PCOS with metabolic syndrome (MS). In addition, the PCOS should be diagnosed following the ESHRE/ASRM recommendation after exclusion of causes of hyperandrogenism such as late onset congenital adrenal hyperplasia (CAH), androgen secreting adrenal or ovarian tumors, and Cushing's syndrome4'5.\nSo, Moustafa et al., should include the late onset CAH, and Cushing`s syndrome in their study exclusion criteria.\nMoustafa et al., mentioned that hypothyroidism, is more common in women with PCOS than in the general population (40% of patients with PCOS showed sub-clinical hypothyroidism (SCH)). Also, they mentioned that the most obvious connection between thyroid diseases, and PCOS seem to be an increase in body mass index (BMI). Enzevaei et al. found that 25.5% of subjects with PCOS have SCH6. Similarly, Sinha et al. found that 22.5% subjects with PCOS were reported to have SCH7.\nThis can be explained by the high BMI of the PCOS-women, which produces relative thyroid hormone deficiency, and SCH. The non-diagnosed SCH of the PCOS-women converted to overt/clinical hypothyroidism with further increase in BMI. Consequently, the overt/clinical hypothyroidism, produces anovulation, and subsequent increased severity of PCOS9. This explains why Abdelazim and Kanshaiym, recommended screening of PCOS-women for the hypothyroidism9.\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": "64726", "date": "29 Jun 2020", "name": "ABM Kamrul Hasan", "expertise": [ "Reviewer Expertise Endocrine disorders", "Diabetes mellitus" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the article, Mayada et al. compared the thyroid hormone profile and other clinical & biochemical parameters of women diagnosed as PCOS with non-PCOS otherwise healthy counterparts. In general, there is a scope of improvement in this article as it is not so well written.\n\nI want to leave the following comments:\n\nThe term ‘polycystic ovary syndrome’ is preferred over ‘polycystic ovarian syndrome’.\nAbstract:\n\n‘The prevalence of polycystic ovaries on ultrasound is around quarter of all women but is not associated with the full syndrome’- is irrelevant here.\n\nThis is not a case-control study, rather a cross-sectional study that involved a comparison group.\n\nIn the result, the comparison of different hormone levels and BMI was done. But, in conclusion, you have commented about the prevalence of thyroid disorders in the two groups.\nIntroduction:\nThe description of the pathophysiological link between PCOS and thyroid disorders is clumsy.\nMethods:\nCross-sectional study, not case-control.\n\nThe sample size is small. How the sample size was calculated?\n\nThe inclusion and exclusion criteria are not clearly defined. Was the PCOS cases treatment naïve?\n\nThe age of the study participants was not mentioned in the method section. The age range of the subjects mentioned in the abstract (13-45 years) is dissimilar to that mentioned in Table 1.\n\nAccording to methodology, TVS was done in all cases. Is it justified for adolescents or sexually inactive (i.e. unmarried in your culture) women?\n\nThe inclusion criteria do not match with the Rotterdam criteria mentioned in the guidelines.\n\nAll PCOS women are not overweight and obese, many of them have normal even low BMI. Why were subjects with normal or low BMI were excluded? Again, patients of these categories are included in the tables. This is confusing.\n\nHyperthyroidism or hypothyroidism are in the exclusion criteria but subjects with normal or low TSH, T4, and T3 are in the tables. Please explain.\n\n‘Good performance status’, what does it mean?\n\nAvoid the confusing and vague terms like ‘Positive past-medical history’, ‘Positive past-surgical history.’ Specify the inclusion and exclusion criteria for both groups.\n\nThe blood collection procedures are not written in detailed. Please mention the methods for the estimation of each hormone.\n\nPlease mention the time of sample collection with respect to the phase of the menstrual cycle and fasting status.\n\nFree T4 and Free T3 were measured according to the method section. But in the tables and result section, you have written T4 and T3. Please explain.\n\nThe measurement of anti-Mullerian Hormone is not mentioned in the method section though it is in the tables. Progesterone is measured according to the method section, it is not mentioned in the result or tables.\n\nSPSS is not appropriately written in the text, please see the website of the manufacturer for correct citation.\nResults:\nThe results are contradictory to the exclusion criteria of the PCOS subjects as I have already mentioned.\n\nTable 1: The ranges should be mentioned in the brackets with the mean values or may be mentioned in the separate columns. FSH is significantly different between the two groups (p=0.007).\n\nTable 2: Were all PCOS women infertile?\n\nThyroid function status (euthyroid or subclinical hypothyroid) was not shown in any table, though in the result section it is written to be included in table 4.\n\nHow many of the comparison group had thyroid dysfunction?\n\nSome suggestions about the presentation of the study result: a) compare all the demographic, clinical, and laboratory parameters between two groups; b) compare the subcategories of the study subjects according to thyroid status, biochemical hyperandrogenism, etc.\nDiscussion:\nThe discussion section needs improvement.\n\nThis part highlighted only the prevalence of SCH in PCOS and just compared the prevalence of this study with the others. There is no explanation regarding the higher prevalence of SCH in PCOS. Anti-thyroid antibodies were not measured in this study though previous findings of thyroid autoimmunity in PCOS were discussed.\n\n‘TSH measures, T4, and T3 may be more applicable in the diagnosis of PCOS.’- I failed to understand this. Thyroid function tests are mandatory to exclude thyroid dysfunctions which sometimes mimic PCOS clinically and radiologically, these are not used to diagnose PCOS.\nConclusion:\nIn the study 60% of the PCOS women were euthyroid, but the authors concluded as ‘most patient with PCOS will have some degree of thyroid dysfunction, especially SCH’, which is not a reflection of the study result.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/8-669
https://f1000research.com/articles/7-1175/v1	02 Aug 18	{ "type": "Case Report", "title": "Case Report: Hepatopulmonary syndrome as the first clinical manifestation of cirrhosis in a patient with underlying chronic lung disease", "authors": [ "Charles Murphy", "Danit Arad", "Charles Murphy" ], "abstract": "An 86 year old woman with multiple chronic lung diseases (including chronic obstructive pulmonary disease, bronchiectasis, and untreated mycobacterium avium-intracellulare) presented with two weeks of increased shortness of breath, notably worse when seated as compared to when lying down. After treatments focused on her known conditions did not resolve her dyspnea, the differential diagnosis was broadened and she was found to have cirrhosis. As a result of this new diagnosis, transthoracic echocardiography and arterial blood gas analysis were performed and together yielded the diagnosis of hepatopulmonary syndrome. We describe a rare presentation of hepatopulmonary syndrome manifesting as a patient’s first clinical evidence of cirrhosis, a diagnosis made difficult by this patient’s numerous other lung diseases which muddied the picture.", "keywords": [ "Hepatopulmonary Syndrome", "COPD", "MAI Infection", "Bronchiectasis" ], "content": "Introduction\n\nThe triad of chronic liver disease, hypoxemia, and microvascular dilatations/malformations in the lungs make up hepatopulmonary syndrome (HPS), a well-known sequela of hepatic cirrhosis1. These aforementioned vascular abnormalities result in intrapulmonary shunting, which yields the clinical findings of dyspnea (universally) as well as digital clubbing and/or cyanosis (both very common)2. We report a case of a patient presenting with HPS as their first clinical manifestation of cirrhosis, and describe how it was distinguishable from other potential causes of her symptoms such as her comorbid chronic obstructive pulmonary disease (COPD), bronchiectasis, and mycobacterium avium-intracellulare (MAI) infection.\n\n\nCase report\n\nAn 86 year old retired latina woman with a past medical history of COPD, bronchiectasis, MAI infection (not previously treated), diabetes mellitus, hyperlipidemia, and hypertension presented with two weeks of worsened dyspnea and non-productive cough. She reported a baseline of daily shortness of breath with an exercise tolerance of 3 blocks, but over the two weeks prior to her presentation it decreased to the point where she would feel dyspneic when walking around her apartment. Interestingly she stated that she also generally felt more short of breath while seated than when lying down, and also cited a worsening cough over this time course productive of green sputum. Her exam on presentation was significant for an oral temperature of 101.4 degrees Fahrenheit, oxygen saturation of 84% on room air, tachypnea and coarse crackles appreciated diffusely on lung examination. Her blood-work was notable for a white blood cell count of 19.8 k/µL, with multiple diffuse small nodular opacities seen on chest x-ray. She was started on levofloxacin for treatment of a presumed bronchiectasis flare along with oxygen therapy via nasal cannula in addition to other supportive treatments. Although her fever, leukocytosis, and cough improved with antibiotics (further supporting a diagnosis of bronchiectasis flare), her dyspnea and hypoxemia persisted. Consequently, a chest computerized tomography (CT) scan was ordered which showed the same extensive nodularities seen on chest x-ray, but also elucidated a nodular liver consistent with cirrhosis. While her platelet count, transaminases, bilirubin, and prothrombin time were all normal and she had no ascites or other edema on exam, she did however have spider angiomas. Further chart review done at that time revealed that she had known cirrhotic characteristics on liver imaging as they were incidentally seen almost five years prior, although she had never had any decompensations or serologic evidence of liver dysfunction since then. Work-up back then elucidated no potential cause except for non-alcoholic fatty liver disease, given her histories of hyperlipidemia and diabetes. In light of this knowledge gained from deep chart review, the specter of hepatopulmonary syndrome was raised as a possible explanation for her persistent hypoxemia and dyspnea. In order to investigate this possibility, both seated and supine arterial blood gases were obtained which elucidated orthodeoxia (see Table 1). A transthoracic echocardiogram with bubble study was then performed which showed an intrapulmonary shunt (see Figure 1), thereby confirming the diagnosis of HPS. While oxygen supplementation caused her dyspnea to improve and oxygen saturation to rise to a safe level, she interestingly was never able to reach a saturation of 100%. However given this improvement in her dyspnea and oxygenation, as well as the resolution of all signs and manifestations of the bronchiectasis flare that she initially presented with, the patient was discharged home with oxygen. Soon after discharge, she was seen in a pulmonology clinic where treatment for MAI was commenced. When last seen a month afterward, the patient reported that she was tolerating her treatments well and felt improved since her hospital discharge.\n\nFiO2– Fraction of inspired oxygen, PaO2– Partial pressure arterial oxygen.\n\nStill images from patient’s transthoracic echocardiogram showing (a) no early shunting with saline bubble (identified by yellow arrows) injection, followed by (b) late passage of bubbles (identified by red arrows) into the Left Atrium and Ventricle representing Intrapulmonary Shunting.\n\n\nDiscussion\n\nOur patient possessed all three of the cardinal findings of hepatopulmonary syndrome: chronic liver disease, hypoxemia, and microvascular dilatations/malformations in the lungs. Her symptom of platypnea (shortness of breath worsened by going from a supine to seated position) and finding of orthodeoxia on arterial blood gas analysis also clearly pointed to HPS. But while most cirrhotic patients with hepatopulonary syndrome have only mild disease and its severity is typically proportional to that of their cirrhosis, she presented with severe disease despite having seemingly compensated cirrhosis3. Moreover, it is very unusual for HPS to be the first symptomatically manifesting sequela of cirrhosis as it was in this patient; there are few other examples of this happening in the literature4.\n\nAs it is classically described, our patient's intrapulmonary vascular abnormalities resulted in intrapulmonary shunting, which yielded her dyspnea and hypoxemia. More specifically, this shunting caused her to have platypnea, which is the symptomatic manifestation of orthodeoxia, and has been shown to be very closely tied with HPS. In one prospective study comparing cirrhotics with HPS vs. those without the complication, platypnea was endorsed by 65.5% of the HPS group vs 6.2% of the non-HPS cirrhotic group5.\n\nGiven that they are also manifestations of cirrhosis-related vascular malformations, spider angiomas are also commonly seen in HPS as they were with our patient6. The intrapulmonary anomalies can be reliably detected via saline-enhanced transthoracic echocardiography, as well as with more advanced confirmatory tests such as technitium-99m macroaggregated albumin (MAA) nuclear scanning or pulmonary angiography3,7,8. Given the overwhelming presence of intrapulmonary shunting seen on her transthoracic echocardiogram though, these more advanced and expensive tests were not pursued in our patient's case.\n\nOnce the diagnosis is made with the aforementioned triad, disease severity is assessed via PaO2. Patients with mild disease have a PaO2 ≥ 80mmHg, those with moderate have PaO2 ≥ 60 < 80 mmHg, severe have PaO2 ≥ 50 < 60 mmHg, and those with very severe disease have a PaO2 of < 50 mmHg9. Our patient was found to have severe disease based upon her PaO2. The pathogenesis is thought to involve increased serum levels of nitric oxide (although correlations with elevated carbon monoxide and tumor necrosis factor α have also been seen) resulting from cirrhosis, which is postulated to cause pulmonary vascular dilatation and arteriovenous malformations (AVMs)7,10. Autopsy studies have shown that the number of dilated precapillary and capillary vessels in the lungs far outnumbers the number of pulmonary AVMs in these patients, but the end result of each is the same: passage of mixed-venous blood into the pulmonary veins, resulting in shunting, V/Q mismatch, and hypoxemia7. Where those vessels dilated as a result of HPS are concerned, the shunting is a result of diffusion-limited gas exchange. Cirrhosis itself (independent of HPS) is also associated with impaired autoregulation of pulmonary vascular tone, and this coupled with the shunting seen in hepatopulmonary syndrome is what is thought to cause orthodeoxia: as gravitational changes in pulmonary blood distribution cannot be accommodated for, there is increased blood supply to the lung bases, where the amount of ventilation relative to perfusion is less than that of the superior lung zones7,11.\n\nThe only definitive treatment for HPS is liver transplantation12. While those with HPS are more prone to post-operative complications than other patients post-liver transplant, ultimately those who survive have resolution of the hypoxemia caused by their pre-transplant HPS13. Given her age, comorbidities, and otherwise well-compensated cirrhosis, liver transplant was not considered in our patient.\n\nIn addition to HPS, there are other similar clinical entities which are worth discussing as part of the differential diagnosis for patients who present like ours. Similar to HPS, portopulmonary hypertension (PPH) is also a sequela of cirrhosis which is characterized by pulmonary hypertension (as defined by elevated mean arterial pressure of >25mmHg at rest, and pulmonary vascular resistance greater than 240 dynes/sec/cm-5) in the setting of portal hypertension and/or cirrhosis with other causes of PH having been excluded9. Patients with PPH typically present with clinical findings consistent with other causes of pulmonary hypertension, namely external dyspnea, fatigue, chest pain, and/or syncope, with progression to cor pulmonale in severe cases14. PPH was excluded in our patient by the fact that her transthoracic echocardiogram showed no evidence of pulmonary hypertension.\n\nHereditary hemorrhagic telangiectasia (HHT) is a genetic (autosomal dominant) disease in which arteriovenous malformations (AVMs) and mucocutaneous telangiectasis form throughout the body15. The predominant symptom with which people present is paroxysmal epistaxis related to AVMs in the nasal mucosa, although 15–35% of patients with HHT have pulmonary AVMs which cause dyspnea in a manner analogous to those with HPS16.\n\nWhile it was tempting at first to presume that our patient's untreated MAI (and/or her other lung diseases, see Table 2 below) could be causing her symptoms, the clinical presentations which result from this infection are not mimickers of HPS. Those with symptomatic pulmonary MAI infection typically present in one of two ways, depending on whether they have underlying lung disease or not. Those with underlying lung disease (most commonly COPD or bronchiectasis) typically have a tuberculosis-like (albeit milder) presentation: chronic cough, weakness, malaise, weight loss, dyspnea, and upper-lobe predominant infiltrates and/or cavitation on chest imaging17. Patients with underlying bronchiectasis will usually have their MAI infection develop in bronchiectatic areas, not necessarily in the upper lobes18. Those without underlying lung disease tend to present with months of productive cough, without the other “tuberculosis-like” constitutional symptoms19. A subset of these patients classically present with “Lady Windermere syndrome:” lingular/right-middle lobe infiltrates in elderly women without predisposing lung disease who suppress their cough20. Regardless of which way a patient presents, diagnosis is made via the combination of radiographic findings indicative of pulmonary disease along with either MAI-positive sputum or an MAI-positive bronchial wash in a patient with respiratory symptoms, according to the Infectious Disease Society of America and the American Thoracic Society21.\n\nCOPD- Chronic obstructive pulmonary disease, MAI - mycobacterium avium-intracellulare infection, HPS – Hepatopulmonary syndrome, PPH – Portopulmonary hypertension, HHT - Hereditary hemorrhagic telangiectasia, WHO - World Health Organization.\n\nHepatopulmonary syndrome belongs on the differential diagnosis for dyspnea in any patient with chronic liver disease, even those with previously compensated cirrhosis like our patient. While it can present in a way which is symptomatically similar to that of other chronic lung diseases, it can be definitively diagnosed via a relatively simple work-up which should be performed in any dyspneic patient with cirrhosis. While the only definitive cure is liver transplant, it can be conservatively managed via oxygen therapy when transplant is contraindicated, as it was in our patient's interesting case.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or 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": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHo V: Current concepts in the management of hepatopulmonary syndrome. Vasc Health Risk Manag. 2008; 4(5): 1035–1041. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammad Alizadeh AH, Fatemi SR, Mirzaee V, et al.: Clinical features of hepatopulmonary syndrome in cirrhotic patients. World J Gastroenterol. 2006; 12(12): 1954–1956. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFragaki M, Sifaki-pistolla D, Samonakis DN, et al.: Screening for Hepatopulmonary Syndrome in Cirrhotic Patients Using Technetium 99m-macroaggregated Albumin Perfusion Lung Scan (Tc-MAA): Diagnostic Approach and Clinical Correlations. J Clin Gastroenterol. 2017. PubMed Abstract \| Publisher Full Text\n\nZieliński M, Hartleb M, Sitek P, et al.: Dyspnoea, cyanosis and digital clubbing in a 28-year-old patient as a result of hepatopulmonary syndrome. Adv Respir Med. 2017; 85(6): 339–344. PubMed Abstract \| Publisher Full Text\n\nYounis I, Sarwar S, Butt Z, et al.: Clinical characteristics, predictors, and survival among patients with hepatopulmonary syndrome. Ann Hepatol. 2015; 14(3): 354–60. PubMed Abstract\n\nSilvério Ade O, Guimarães DC, Elias LF, et al.: Are the spider angiomas skin markers of hepatopulmonary syndrome? Arq Gastroenterol. 2013; 50(3): 175–179. PubMed Abstract \| Publisher Full Text\n\nRodríguez-Roisin R, Krowka MJ: Hepatopulmonary syndrome--a liver-induced lung vascular disorder. N Engl J Med. 2008; 358(22): 2378–2387. PubMed Abstract \| Publisher Full Text\n\nOffer J, Green L, Houghton AR, et al.: A case of hepatopulmonary syndrome. Echo Res Pract. 2015; 2(2): K25–K27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodríguez-roisin R, Krowka MJ, Hervé P, et al.: Pulmonary-Hepatic vascular Disorders (PHD). Eur Respir J. 2004; 24(5): 861–80. PubMed Abstract \| Publisher Full Text\n\nCremona G, Higenbottam TW, Mayoral V, et al.: Elevated exhaled nitric oxide in patients with hepatopulmonary syndrome. Eur Respir J. 1995; 8(11): 1883–1885. PubMed Abstract \| Publisher Full Text\n\nGómez FP, Martínez-Pallí G, Barberà JA, et al.: Gas exchange mechanism of orthodeoxia in hepatopulmonary syndrome. Hepatology. 2004; 40(3): 660–6. PubMed Abstract \| Publisher Full Text\n\nRodriguez-Roisin R, Krowka MJ: Is severe arterial hypoxaemia due to hepatic disease an indication for liver transplantation? A new therapeutic approach. Eur Respir J. 1994; 7(5): 839–842. PubMed Abstract\n\nEriksson LS, Soderman C, Ericzon BG, et al.: Normalization of ventilation/perfusion relationships after liver transplantation in patients with decompensated cirrhosis: evidence for a hepatopulmonary syndrome. Hepatology. 1990; 12(6): 1350–1357. PubMed Abstract \| Publisher Full Text\n\nHoeper MM, Krowka MJ, Strassburg CP: Portopulmonary hypertension and hepatopulmonary syndrome. Lancet. 2004; 363(9419): 1461–8. PubMed Abstract \| Publisher Full Text\n\nSharathkumar AA, Shapiro A: Hereditary haemorrhagic telangiectasia. Haemophilia. 2008; 14(6): 1269–80. PubMed Abstract \| Publisher Full Text\n\nKrynytska I, Marushchak M, Mikolenko A, et al.: Differential diagnosis of hepatopulmonary syndrome (HPS): Portopulmonary hypertension (PPH) and hereditary hemorrhagic telangiectasia (HHT). Bosn J Basic Med Sci. 2017; 17(4): 276–285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeirstein AS, Damsker B, Kirschner PA, et al.: Pulmonary infection with Mycobacterium avium-intracellulare: diagnosis, clinical patterns, treatment. Mt Sinai J Med. 1990; 57(4): 209–15. PubMed Abstract\n\nKilby JM, Gilligan PH, Yankaskas JR, et al.: Nontuberculous mycobacteria in adult patients with cystic fibrosis. Chest. 1992; 102(1): 70–5. PubMed Abstract \| Publisher Full Text\n\nPrince DS, Peterson DD, Steiner RM, et al.: Infection with Mycobacterium avium complex in patients without predisposing conditions. N Engl J Med. 1989; 321(13): 863–8. PubMed Abstract \| Publisher Full Text\n\nReich JM, Johnson RE: Mycobacterium avium complex pulmonary disease presenting as an isolated lingular or middle lobe pattern. The Lady Windermere syndrome. Chest. 1992; 101(6): 1605–9. PubMed Abstract \| Publisher Full Text\n\nGriffith DE, Aksamit T, Brown-Elliott BA, et al.: An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007; 175(4): 367–416. PubMed Abstract \| Publisher Full Text" }	[ { "id": "37169", "date": "29 Aug 2018", "name": "Robert Rodríguez-Roisin", "expertise": [ "Reviewer Expertise COPD and hepatic vascular disorders" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is another case report of an interesting and complex HPS in the context of several comorbidities, such as COPD and bronchiectasis, let alone the coexistence of MAI infection, that each, all in combination or even one more than another entity, can cause arterial hypoxaemia, along with an incomplete differential diagnosis approach. All in all both the discussion and presentation are weak and modest which detract from the potential clinical interest of the case report. Behind this negative short review lie three major caveats.\nFirst, the lack of a better knowledge on gas exchange abnormalities in HPS is overwhelming despite that some of the quoted references (i.e., ref 7) masterly describe the pathophysiology of gas exchange alterations in HPS. Yet, the Authors miss a succinct good description. One of the reasons of this is that the Author is confounded by the use of the broad term of ‘shunting’ to encompass the three well established intrapulmonary determinants of arterial hypoxaemia in HPS (ventilation-perfusion imbalance, increased intrapulmonary shunt, and diffusion limitation to O2 transfer) quite thoroughly reported in a recent publication of the Amer Physiol Soc (Rodriguez-Roisin R, et al.1). May I therefore suggest to the Author to take a look at this publication and make the appropriate changes in your paper. HPS is not a problem of ‘intrapulmonary shunting’, as it is repeatedly quoted, but a predominant ventilation-perfusion mismatching along with other additional altered pulmonary and non-pulmonary determinants of gas exchange (see also, Rodriguez-Roisin2).\nSecond, the Author does not afford the lung function testing of this lady, including the most likely smoking habits of his old patient which are completely ignored. As a result, we are in the middle of nowhere to grasp the comprehensive nature of her gas exchange abnormalities. Are these due to her severe chronic pulmonary problems (COPD with bronchiectasis), to HPS as the author is inclined to, without providing any rationale, or to a combination of both pulmonary and liver disorders (most likely). The lack of a proper differential diagnosis is another of the main gaps. I, for me, suggest to also read a very interesting paper focusing on cardiopulmonary comorbidities and HPS (Martinez GP, et al.3) that may help the Author to improve his differential diagnosis. Moreover, the Author has completely neglected the relevant role of the alveolar-arterial oxygen partial pressure difference. With due respect, I suggest you read again ref 7, now more critically and more in detail!\nThird, I have to admit that although it is most likely that the arterial hypoxaemia of this old lady is due to a combination of lung and hepatic problems, there is a sentence in the Case Report that worries me. It says: ‘Soon after discharge, she was seen in a pulmonology clinic where treatment for MAI was commenced. When last seen a month afterward, the patient reported that she was tolerating her treatments well and felt improved since her hospital discharge’. This sentence makes me suspicious and questions me if the hypoxaemia is due to HPS for there are no pharmacological options for HPS so far.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/7-1175
https://f1000research.com/articles/8-667/v1	15 May 19	{ "type": "Case Report", "title": "Case Report: Concomitant coronary stent and femoral artery thrombosis in the setting of heparin-induced thrombocytopenia", "authors": [ "Mejdi Ben Messaoud", "Mezri Maatouk", "Mohamed Mehdi Boussaada", "Marouane Mahjoub", "Walid Mnari", "Habib Gamra", "Mezri Maatouk", "Mohamed Mehdi Boussaada", "Marouane Mahjoub", "Walid Mnari", "Habib Gamra" ], "abstract": "Heparin induced thrombocytopenia (HIT) is a rare but potentially life threatening adverse drug reaction. We report an unusual case of concomitant subacute coronary stent and femoral artery thrombosis secondary to HIT. In the current era of extensive growth of heparin use and percutaneous coronary interventions, it’s important for clinicians to remember that such complication might occur and should be prevented.", "keywords": [ "Key words: heparin", "thrombocytopenia", "thrombosis", "coronary artery", "stent", "myocardial infarction", "femoral artery", "coronary angioplasty", "thrombectomy" ], "content": "Introduction\n\nHeparin is a commonly used anticoagulant for hospitalized patients, but its use can lead to devastating complications, such as heparin-induced thrombocytopenia (HIT)1. We report an unusual case of concomitant subacute coronary stent and femoral artery thrombosis in the setting of HIT. This case highlights the importance of considering HIT as a cause of coronary or arterial thrombosis few days after heparin exposure.\n\n\nCase report\n\nA 66-year-old male patient, with a history of smoking (30 pack-years) and no known medical or surgical history, was admitted in our department for a spontaneously resolved inferior ST elevation myocardial infarction (STEMI). The intra-hospital treatment included enoxaparin 0.6 ml twice a day, clopidogrel 75 mg once a day, aspirin 100 mg once a day, bisoprolol 2.5 mg once a day and atorvastatin 40 mg once a day. The coronary angiogram (performed at day 3 through the right radial artery) showed a severe thrombotic lesion of the distal circumflex. The patient underwent an ad-hoc successful angioplasty of the circumflex with a drug eluting (everolimus) stent. Initial laboratory tests at admission were normal except elevated troponin. Echocardiography showed a 65% left ventricular ejection fraction. The patient was discharged after 5 days of anticoagulation by low molecular weight heparin (enoxaparin). Laboratory tests were not controlled during the hospitalization. The discharge treatment included clopidogrel 75 mg once a day, aspirin 100 mg once a day, bisoprolol 2.5 mg once a day and atorvastatin 40 mg once a day.\n\nOne week later, the patient was referred again to our department for both chest and right lower limb pain. The electrocardiogram showed an inferior STEMI and the physical exam of the right lower limb found ischemic signs with absence of the femoral pulse. There was no history of aspirin or clopidogrel discontinuation. An urgent coronary angiogram (performed through the left femoral artery) showed total thrombosis of the circumflex stent (Figure 1A). The patient underwent a successful primary angioplasty of the circumflex by simple balloon (Figure 1B). Urgent lower limb contrast-enhanced computed tomography was performed immediately after the angioplasty, revealing total acute thrombosis of the right common femoral artery (Figure 2A, B). The patient underwent an urgent successful thrombectomy with Fogarty catheter. Immediate evolution was favorable with total regression of coronary and right lower limb ischemic signs. Laboratory tests showed a marked fall in the platelet count (68,000/L) which was normal (364,000/L) in the previous hospitalization. A diagnosis of concomitant coronary stent and femoral artery thrombosis due to HIT was strongly suspected (4T score = 8). Our therapeutic strategy was immediate discontinuation of low molecular weight heparin (enoxaparin), aspirin and clopidogrel with strict daily control of platelet count. During this period, no alternative anticoagulation was initiated because of the unavailability of direct thrombin inhibitors in our center. Anticoagulation with a vitamin K antagonist (acenocoumarol 4 mg once a day) and dual antiplatelet therapy with aspirin 100 mg once a day and clopidogrel 75 mg once a day were initiated at day 3 once platelet count had recovered. The in-hospital outcome was favorable and the patient was discharged after 15 days on acenocoumarol 4 mg once a day, aspirin 100 mg once a day and clopidogrel 75 mg once a day. The 3-month follow-up, with controlled blood tests and lower limb contrast-enhanced computed tomography showing total reperfusion of the right femoral artery (Figure 2C), was unremarkable.\n\n(A) The intra-stent thrombosis of the circumflex coronary artery. (B) The final result after successful angioplasty of the circumflex coronary artery.\n\n(A, B) show the thrombotic occlusion of the right common femoral artery. (C) Reperfusion of the right femoral artery after thrombectomy.\n\n\nDiscussion\n\nHIT is defined as a sudden fall in the platelet count (e.g. <100,000 per L or >50% drop from baseline) a few days after heparin use. Its incidence ranges from <1% to 7% depending on the heparin type (more than 10 times higher with unfractionated heparin compared to low-molecular-weight heparin), duration of heparin exposure and patient population1. There are two types of HIT, with type I HIT the most common form. Type 1 HIT is due to the direct effect of heparin on platelets and may manifest as only a slight decrease in platelet count, mostly within 2 days of commencing heparin. Type II HIT is secondary to the formation of antibodies against the heparin-platelet factor 4 complex, resulting in 50% of cases in thrombotic events, mostly in veins, within 5 to 14 days. Coronary artery thrombosis secondary to HIT is very rare and usually occurs in the setting of coronary stents or bypass grafts2. However, the concomitant occurrence of coronary stent and other arterial site thrombosis secondary to HIT is very rare and few cases have been reported in the literature3. The use of scoring systems such as the “4T score” is helpful in assessing the pretest probability of HIT4. Platelet factor 4–heparin antibody tests should be ordered only if the diagnosis of HIT is strongly suggested by clinical features5.\n\nTreatment of HIT requires immediate discontinuation of all heparin products and initiation of alternative therapeutic dose anticoagulation, including direct thrombin inhibitors (argotraban, bivalirudin, fondaparinux, danaparoid) or direct oral anticoagulants (apixaban or rivaroxaban or dabigatran)6. The decision to continue antiplatelet therapy during treatment with a non-heparin anticoagulant may be influenced by the risk of vascular events and bleeding. Routine platelet transfusion is not recommended for patients with acute HIT and thrombosis or average bleeding risk, but it may be an option for patients with active bleeding or at high bleeding risk. In the acute phase of HIT with life-threatening thrombosis, bivalirudin (or argatroban if bivalirudin is unavailable) is the best option for alternative anticoagulation therapy7. Primary angioplasty and thrombectomy with Fogarty catheter should be recommended in the setting of life-threatening thrombosis with STEMI or acute limb thromboembolism8. Oral anticoagulation is required for at least 3 months, preferably with direct oral anticoagulant, which can be initiated at the first day. Warfarin is the most highly recommended vitamin K antagonist when indicated, and should not be given until platelets have substantially recovered (e.g. usually to at least 150 000 per L)7. In our case, acenocoumarol was the sole available alternative anticoagulation therapy. It was initiated at day 3 after platelet count recovery and was continued for 3 months.\n\n\nConclusion\n\nConcomitant coronary and femoral artery thrombosis due to HIT is a rare life-threatening complication of heparin therapy. The present case highlights the importance of considering such diagnosis among patients with prior heparin exposure. Prompt identification and management of this disorder is critically important to avoid devastating complications. To prevent such events, strict control of platelet count during heparin therapy is of paramount importance.\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": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMartel N, Lee J, Wells PS: Risk for heparin-induced thrombocytopenia with unfractionated and low-molecular-weight heparin thromboprophylaxis: a meta-analysis. Blood. 2005; 106(8): 2710–5. PubMed Abstract \| Publisher Full Text\n\nShin HW, Yoon HJ, Choi SW, et al.: Acute Stent Thrombosis and Heparin Induced Thrombocytopenia in a Patient With ST-Segment Elevation Myocardial Infarction. Korean Circ J. 2012; 42(9): 646–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuzuki H, Tsunematsu T, Takahashi H, et al.: A case of heparin-induced thrombocytopenia with subacute stent thrombosis, multiple cerebral infarction, and acute limb ischemia. J Cardiol Cases. 2017; 15(5): 145–149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWarkentin TE: Think of HIT. Hematology Am Soc Hematol Educ Program. 2006; 408–14. PubMed Abstract \| Publisher Full Text\n\nGreinacher A: CLINICAL PRACTICE. Heparin-Induced Thrombocytopenia. N Engl J Med. 2015; 373(3): 252–61. PubMed Abstract \| Publisher Full Text\n\nWarkentin TE, Pai M, Linkins LA: Direct oral anticoagulants for treatment of HIT: update of Hamilton experience and literature review. Blood. 2017; 130(9): 1104–1113. PubMed Abstract \| Publisher Full Text\n\nCuker A, Arepally GM, Chong BH, et al.: American Society of Hematology 2018 guidelines for management of venous thromboembolism: heparin-induced thrombocytopenia. Blood Adv. 2018; 2(22): 3360–3392. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson JL, Morrow DA: Acute Myocardial Infarction. N Engl J Med. 2017; 376(21): 2053–2064. PubMed Abstract \| Publisher Full Text" }	[ { "id": "48571", "date": "04 Jun 2019", "name": "Batric Popovic", "expertise": [ "Reviewer Expertise Thrombotic disorders and interventional cardiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments:\n\nI read with great interest the report written by Mejdi Ben Messaoud et al. about an unusual case of concomitant subacute coronary stent and femoral artery thrombosis secondary to heparin induced thrombocytopenia. There had been previous reported cases of HIT causing coronary thrombus formation but not with concomitant extracoronary thrombotic events.\n\nThe authors confirm that a heparin therapy needs a platelet count at least 2/ week which is a well-known recommendation. I think that authors should also emphasize that a prolonged and unnecessary anticoagulation after percutaneous coronary intervention may be deleterious. Indeed, different studies reported that in elective PCI or after primary angioplasty after myocardial infarction, the anti coagulation should be early discontinued.\n\nThis report needs several minor modifications as follows:\n\nAbstract:\n\n“In the current era of extensive growth of heparin use and percutaneous coronary interventions, it’s important for clinicians to remember that such complication might occur and should be prevented.” This sentence should be modified: PCI required perprocedural anticoagulation but dose and duration of anticoagulation have been decreased last decade. (See: Montalescot STEEPLE investigators and Futura/OASIS 8).\n\nCase report:\n“A 66-year-old male patient, with a history of smoking (30 pack-years) and no known medical or surgical history:”. 30 pack years should be deleted and “no known”: means without?\n\nEchocardiography showed a 65% left ventricular ejection fraction. The authors should specify the presence or not of an intra cardiac mass. Indeed, one of the most frequent underlying cardiac diseases concomitant coronary and extra coronary acute thrombotic events is intracardiac mass/tumor.\n\nThe in-hospital outcome was favorable and the patient was discharged after 15 days on acenocoumarol 4 mg once a day, aspirin 100 mg once a day and clopidogrel 75 mg once a day. Indicate the duration of this antithrombotic strategy in this paragraph and not in the discussion.\n\n“The 3-month follow-up, with controlled blood tests and lower limb contrast-enhanced computed tomography showing total reperfusion of the right femoral artery ,was unremarkable.” The sentence may be modify: “At 3 month follow up, no recurrent thrombotic events occurred, controlled blood tests were normalized and low limb contrast-enhanced computed tomography completely reperfused.\"\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "49698", "date": "04 Jul 2019", "name": "Marouane Boukhris", "expertise": [ "Reviewer Expertise Interventional cardiology" ], "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 current manuscript, the authors reported the case of a STEMI patient who suffered from heparin-induced thrombocytopenia resulting not only in stent thrombosis but also in femoral artery thrombotic occlusion. Overall, the paper is well written and of interest for the reader. It is suitable for indexing in its actual version.\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/8-667
https://f1000research.com/articles/8-448/v1	11 Apr 19	{ "type": "Research Article", "title": "The association between health literacy and pesticide use behaviors among sweet corn farmers in the Pak Chong district of Thailand: a cross-sectional study", "authors": [ "Theerachai Pobhirun", "Somdej Pinitsoontorn", "Theerachai Pobhirun" ], "abstract": "Background: Pesticide toxicity is an important health problem in Thailand due to the intensive use of hazardous pesticides. This study aimed to determine and discuss patterns of pesticide use, health literacy, pesticide use behaviors and whether there is an association between health literacy and pesticide use behaviors among sweet corn farmers in the Pak Chong district of Thailand. Methods: This work was carried out between May 2017 and July 2017 and 161 participants were enrolled. Participant questionnaires were completed during face-to-face interviews. Results: 161 sweet corn farmers (89.98%) were interviewed about patterns of chemical pesticide use. Two of the pesticides used in the pre-planting phase were moderately toxic: paraquat (used by 55.2% of farmers) and imidacloprid (used by 15.5% of farmers). In the pre-emergence phase, participants reported using two moderately toxic pesticides: alachlor (used by 48.8% of farmers) and chlorpyrifos (used by 2.4% of farmers). At the post-emergence phase, participants reported using six moderately toxic pesticides: chlorpyrifos (used by 60.7% of farmers), paraquat (used by 38.1% of farmers), imidacloprid (used by 7.2% of farmers), 2-4D (used by 3.6% of farmers), abamectin (used by 3.6% of farmers) and cypermethrin (used by 1.2% of farmers). Health literacy levels were moderate level (Mean score = 91.62, SD = ± 7.06) and pesticide use behaviors were low level (Mean score = 67.80, SD = ± 4.04). When examining the association between health literacy and pesticide use behaviors, we found that functional literacy was significantly associated with pesticide use behaviors. These findings suggest that health literacy, which includes self-management and decision-making skills, should be given greater attention as pesticide use behaviors were unsafe. Conclusion: It may be necessary to develop approaches to reduce pesticide use and promote health literacy, thereby protecting farmers, consumers, the environment (soil, water, and air) and ecosystems from pesticide-related hazards.", "keywords": [ "Pesticides", "Health literacy", "Sweet corn", "Harm reduction", "Farmers", "Pattern", "Behavior" ], "content": "Introduction\n\nAgricultural production accounts for 10% of the Kingdom of Thailand’s gross domestic product (GDP) and 60% of employment. As in many developing countries, in order to achieve high yields, farmers in Thailand use pesticides and herbicides to control pests, weeds and other pathogens. Despite following stricter regulations, increasing evidence of the health risks associated with exposure to pesticides and pesticide residues contaminating food, water and air1,2, imports of insecticides, herbicides, and fungicides have peaked in the last decade, particularly between 2011 to 20173. The top ten pesticides imported by Thailand are glyphosate, paraquat, 2,4-D-dimethylammonium, atrazine, ametryn, 2,4-D-sodium, diuron, propyl, chlorpyrifos, and mancozeb4. The World Health Organization (WHO) classification of pesticides by hazard has been aligned with the GHS (The Globally Harmonized System of Classification and Labelling of Chemicals) acute toxicity hazard categories: Ia = extremely hazardous; Ib = highly hazardous; II = moderately hazardous; III = slightly hazardous; U = unlikely to present an acute hazard; O = Obsolete as pesticide (not classified)3. Those pesticides classified as toxic include glyphosate (III), paraquat (II), 2,4-D-dimethylammonium (II), atrazine (III), ametryn (II), 2,4-D-sodium, diuron (III), propyl (O), chlorpyrifos (II), and mancozeb (U). Currently, the Thai Pesticide Alert Network (Thai-PAN) is campaigning and working with the government of Thailand to ban four pesticides (paraquat, glyphosate, chlorpyrifos, and 2,4D) due to their long-term health effects on humans. In addition, Thailand has adopted the Sufficiency Economy Philosophy as a guideline for agriculture. According to this philosophy, building immunity to environmental changes prompts individuals and their communities to be aware of the impacts their actions may have on the environment and, subsequently, their livelihoods, an awareness which leads them to live in harmony with nature5. The environmental aspects of this guideline enable people and communities to realize the effects of pesticides on health, environment and livelihoods.\n\nHowever, pesticide use trends indicate that pesticide use is increasing6. Although pesticides are beneficial for the control of pests, there are serious concerns for humans and animals regarding associated health risks. Pesticides are chemicals which have been designed to be toxic to pests and, in many cases, their toxic nature can also be harmful to human health1,7,8 and cause environmental pollution9. There have been reports of strong associations between exposure to pesticides and cancer10, and studies have shown that the exposure of pregnant women and breastfeeding mothers to pesticides is a risk factor for low birth weight, congenital birth defects, growth defects, learning disabilities and miscarriage1,11. Previous evidence suggests that farmers have awareness and a basic understanding of insecticides, pesticides and various other chemicals used in farming practices12–14 However, this awareness and knowledge alone might not lead to reduced use of the various chemicals when farming. Health literacy (HL) is a relatively new concept for social and medical sciences research. HL is the ability of individuals to understand basic health information and gain access to essential services, thereby enabling them to make informed decisions9,10. Health literacy can be separated into three levels15–17: (1) Functional literacy, which focuses on the ability to read, understand and access pesticide information; (2) Interactive literacy, which involves the use of cognitive skills and operates in a social environment that supports social participation in health-related issues in the community; (3) Critical literacy, which is the ability to evaluate health issues and determine the challenges of these issues.\n\nThe patterns of pesticide use were studied in three phases of sweet corn farming: pre-planting, pre-emergence and post-emergence. Sweet corn farmers use various methods when applying pesticide such as mixing, loading, spraying and washing equipment. Mixing involves weighing or measuring the pesticide in some fashion and mixing the measured concentrated product with a diluent. Loading involves pouring the diluted pesticide into the spray equipment. Spraying involves the application of the pesticide to control pests by spraying. This is the most common activity for farmers. Typical equipment used may be backpack sprayers or hand-held tank sprayers. Washing involves cleaning the equipment used for pesticide application which may be contaminated during the spraying operation. Pesticide use patterns included the number, type, toxicity and concentration of pesticides used and pesticide use behaviors. There are very few studies about pesticide use among sweet corn farmers. The aim of this research was to determine and discuss patterns of pesticide use, HL of pesticides used, pesticide use behaviors and the association between HL and the behaviors of sweet corn farmers.\n\n\nMethods\n\nThis research applied a cross-sectional, quantitative design to identify determinants of health literacy and pesticide use. This study was carried out from May 2017 to July 2017. The participants were sweet corn farmers in the Pak Chong district of Nakhon Ratchasima province, Thailand. Specifically, the study was conducted in the Klangdong, Chanthuek, Wang Sai and Mu Si sub-districts of the Pak Chong district as these are the regions in which sweet corn is grown. Within these regions, 194 sweet corn farmers were identified through visits to the farms in the area. However, 33 farmers were unwilling to be interviewed.\n\nThe research framework was approved by the ethical committee of Khon Kaen University, Thailand (HE601107). The eligibility criteria of the respondents include sweet corn farmers who live in Pak Chong district, who had used a pesticide for at least 6 months, who could read and write and who were willing to participate in the study. Written informed consent was obtained from all the participants (n = 161). Interviews were conducted by the author (T.P.) using a questionnaire, which included questions about socio-demographic factors and patterns of pesticide use, and answers from farmers were given orally. In order to assess health literacy, the three levels were further separated into 6 dimensions: cognitive skills, access, communication skills, self-management, media literacy and decision-making skills. Cognitive skills refers to the knowledge and understanding the participant has about the correct use of pesticides. Access refers to the access the participant has to pesticide effect information and health services. Communication skills refers to the ability of the participant to listen, speak, read and write about pesticide use. Self-management refers to the ability of the participant to protect their health and reduce the impact on the environment of pesticide application. Media literacy refers to the ability to compare information regarding pesticide efficacy from media such as newspapers, radio, television and pesticide advertisements. Decision-making skills refers to the ability of the participant to decide what pesticides to use and how to use them in a way that does not have a negative impact on their health or the environment.\n\nThree experts from Thai-PAN (Thai Pesticide Alert Network) verified the content validity. The consistency index for all items was 0.61. A pilot test (sample size of 30) was used to achieve clarity for the questionnaires. Cronbach’s alpha, a measure of internal consistency, was 0.72 for the HL of pesticide use section of the questionnaire and 0.77 for the pesticide use behaviors section. Responses to questions 1–10 (cognitive skills) were marked as correct or incorrect. Responses to the remaining questions were scored qualitatively, using a 5-point Likert scale. The item responses ranged from 1 (very low) to 5 (very high). The HL of pesticide use section was composed of 36 items and it was separated into 6 parts (access, cognitive skills, communication skills, self-management, media literacy and decision-making skills). Responses to the 36 items were scored as low, moderate or high levels of health literacy for pesticide use. The maximum score for health literacy was 135. A score of <81 was considered to be low, a score of 81–108 was considered to be moderate and a score of >107 was considered to be high. A breakdown of the score thresholds for each dimension and level of health literacy can be found in Table 1. The pesticide use behaviors section was composed of 20 items, divided into 3 parts (before application, pesticide application and after application). Responses to the 20 items were scored as low, moderate and high standards of pesticide use behaviors. The maximum score for pesticide use behaviors was 100. A score of <67 was considered to be low, a score of 67–69 was considered to be moderate and a score of >69 was considered to be high. A breakdown of the score thresholds for each phase of pesticide application can be found in Table 2.\n\nDescriptive statistics, such as frequencies, percentage, mean and standard deviation (S.D.) are used to report the socio-demographic factors, number of years using pesticides, number of annual pesticide applications, total types of pesticide used in pre-planting, pre-emergence and post-emergence phases of sweet corn farming. In addition, pesticide use behaviors such as; the type of pesticides, the level of toxicity of pesticides and the concentration of pesticides are reported using descriptive statistics. The Chi-squared test was used to test the association between HL and pesticide use behaviors using SPSS (version 20). This analysis could also be performed using a non-proprietary software such as R.\n\nDuring the data collection stage, we had participants provide their written consent. Personal identifiers (names, full addresses) were stripped from the dataset. This research project was approved by the Human Research Ethical Committee of Khon Kaen University (HE601107) based on the principles of the Declaration of Helsinki and the Good Clinical Practice standards of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).\n\n\nResults\n\nThe majority (54%) of sweet corn farmers were male and between 41–60-years-old (mean 43.14 ± 9.97 years). The average farm size for sweet corn planting was 4.67 acres (min = 1.18, max = 15.81 acres). Most of the participants had been using pesticides for between 6 and 10 years (mean 9.04 ± 4.61 years). Most (64.3%) farmers reported planting two sweet corn crops per year. The two pesticide application methods reported were spraying (reported by 88.8% of farmers) and mixing (reported by 83.2% of farmers).\n\nPatterns of pesticide use were studied over three phases of sweet corn farming. First, the soil is prepared for planting (pre-planting phase). 52.2% of farmers reported spraying paraquat to control broadleaf weeds and 16.7% of farmers reported following this with glyphosate, also to control weeds. Paraquat is a class II toxin and glyphosate is a class III toxin. According to the WHO standard and International Agency for Research on Cancer (IARC), glyphosate is likely carcinogenic to humans. For protection against insect pests, 15.5% of farmers mixed corn seeds with imidacloprid before planting, which is also a class II toxin according to the WHO. In addition, 3.6% of farmers used atrazine to control broadleaf weeds in this phase. In summary, four pesticide types were used during the pre-planning phase, of which two are considered to be class II toxins.\n\nDuring the pre-emergence phase, three pesticide types were used: alachlor spray (used by 48.8% of farmers) to control broadleaf weeds, chlorpyrifos spray (used by 2.4% of farmers) to eliminate some pests including insects and worms and atrazine (used by 38.1% of farmers) to control broadleaf weeds. Both alachlor and chlorpyrifos are toxic (class II) according to the WHO. In summary, during the pre-emergence phase, two types of pesticides were class II.\n\nDuring the post-emergence phase, pesticides and herbicides are used to eliminate insect pests and weeds. Chlorpyrifos was used by 60.7% of farmers, imidacloprid by 7.2% of farmers, abamectin by 3.6% of farmers and cypermethrin by 1.2% of farmers to eliminate insect pests, all of which are class II toxins. Paraquat (used by 38.1% of farmers) and 2,4D (used by 3.6% of farmers) were used to eliminate weeds, both of which are class II toxins. Atrazine and glyphosate (both used by 2.4% of farmers) belong to toxicity class III (Table 3). The International Agency for Research on Cancer (IARC) has defined glyphosate and 2,4D (but not atrazine) as carcinogenic.\n\nWe assessed the sweet corn farmers’ level of HL regarding pesticide use. The majority (91.30%) of the sweet corn farmers enrolled in this study had a moderate level of HL (Table 4). We found that the cognitive score was mostly (54.66% of farmers) low, access to information was mostly (77.64% of farmers) moderate, communication skills were mostly (96.89% of farmers) low, self-management was mostly (95.65% of farmers) high, media literacy was mostly (74.53% of farmers) low and decision-making skills were moderate for 42.86% of participants and low for 41.61%. When HL was divided into the three levels, functional HL for the majority of participants (53.42%) was moderate and was classed as high for 45.34%. Interactive HL was low for 9.94% and moderate for 87.58% of participants. Critical HL was low for 53.42% and moderate for 45.34% of participants. The overall analysis of pesticide use behaviors found that 37.27 % of farmers had a low standard of pesticide use behaviors and 31.68% of farmers had a moderate standard of pesticide use behaviors. When we analyzed the three phases of pesticide use (1. before pesticide application, 2. pesticide application, 3. pesticide application), we found that 44.10% of sweet corn farmers had a moderate standard of pesticide use behaviors at the ‘before pesticide application’ phase (mean score 14.2, ± 1.51), 42.24% had a low standard of pesticide use behaviors at the ‘application’ phase (mean score 33.16, ± 2.71) and 43.48% of farmers had a moderate standard of pesticide use behaviors at the ‘after application’ phase (mean score 19.91, ± 1.22) (Table 5).\n\nThe results of the data analysis showed that there was a significant statistical association between health literacy level and pesticide use behaviors (p < 0.05). Of the sweet corn farmers who had a moderate overall health literacy level, 33.33% were inclined towards a high standard of pesticide use behaviors. We found that there was an association between the self-management and decision-making skills dimensions of health literacy and pesticide use behaviors (p < 0.01). Of the sweet corn farmers who had a moderate level of self-management and a high level of decision-making skills, 85.71% and 48.00% were inclined to high standard of pesticide use behaviors, respectively. Regarding functional literacy, there was a significant statistical association with pesticide use behaviors (p < 0.01). Of the sweet corn farmers who had a moderate level of functional health literacy, 39.53% were inclined towards a high standard of pesticide use behaviors. (Table 6).\n\nPercentages represent the proportion of farmers with a particular level of HL who also have a particular standard of pesticide use behaviors. The p-value for association between health literacy and behavior, using the probability exact test. (p<0.05,*p<0.01).\n\n\nDiscussion\n\nFindings of patterns of pesticide use, health literacy and pesticide use behaviors suggest that it is necessary to reduce the harmful effects of pesticide use. Findings included (1) the majority of sweet corn farmers did not follow the current recommended dose or number of sprays, (2) there are many pesticides used in the planting of sweet corn, (3) pesticides used in growing sweet corn have moderate toxicity and (4) the pesticide use behaviors during all phases of planting are unsafe18. However, assessing the risk of exposure to pesticides for humans is not easy because of differences in the duration and levels of exposure to the substance2,7. In addition, the level of HL of the farmers was moderate. Based on these findings, pesticide use by these farmers may affect environmental health8,9 and lead to the pollution of soil and water (surface and groundwater) and diseases of humans19.\n\nTherefore, training sweet corn farmers to reduce the use of pesticides20,21 through an intervention program may be necessary to improve farmer safety. These findings are also consistent with previous studies22,23 regarding health literacy (HL) and pesticide use behaviors. When the sweet corn farmers have knowledge of the pesticide being used, they may have more appropriate pesticide use behaviors. This leads to the suggestion of developing guidelines for appropriate pesticide use for each plant, following a study of the types of pesticides used for each species.\n\nDeveloping HL of the risks of pesticide use requires effective communication in order to lead to an improvement in pesticide use by sweet corn farmers, which could lead to the improvement in health in this century24. There are a number of ways to reduce the harmful effects of pesticide use. (1) Education for farmers about how to use PPE17,21,25 with a focus on apron, mask and wide-brimmed hat use because the relative absorption rate of pesticides is highest in the genital area, ear canal, forehead and scalp (11.8% 5.4% 4.2% and 3.7%)26. Moreover, product labels should be developed that are easy to understand. (2) Promote the use of PPE while mixing, loading and applying any pesticides, especially aprons and goggles or face shields. (3) Behavior such as eating, drinking water or alcohol and smoking should not be undertaken in areas where spraying of pesticides is necessary because the body can absorb the pesticides. (4) Farmers should take a bath at the farm to avoid contaminating family or close friends27. However, farmers cannot bathe in the immediate area where they use pesticides. There are two ways to do this: use a water jug for bathing or change clothes immediately before returning home, separating the pesticide-contaminated clothing from other clothes. (5) Sweet corn planting farmers use up to nine pesticides, seven of which belong to toxicity class II according to the WHO, including 2-4D, glyphosate and atrazine that likely cause cancer14. Sweet corn farmers should be careful with pesticide use, should have access to information regarding pesticide toxicity and should be encouraged to switch to organic farming methods. (6) The harm reduction principle should be applied to promote a reduction in the number of pesticides used, reduce the use of especially toxic pesticides and promote the use of the recommended pesticide concentrations. This would help to protect sweet corn farmers, others indirectly exposed to the pesticides, consumers and the environment. However, promoting the use of organic farming might be rejected by farmers as some sweet corn farmers prefer to use unsuitable pesticides in order to ensure yield and quality28.\n\nThis study was successful in collecting data with a high response rate of 82.98%. The findings of the present study can also be applied to other similar contexts. The authors suggest further qualitative study on the beliefs of farmers regarding pesticide use and organic farming as well as the study of pesticide risk reduction programs. The health literacy questionnaire was based on previously published work, pre-tested, pilot tested and edited to ensure accurate translation, coherence and relevance. However, patterns of pesticide use may be better measured using open-ended questions, where farmers are asked to provide information freely without being influenced.\n\n\nConclusions\n\nThe present study found that the health literacy of pesticide use in sweet corn farmers was at a moderate level and pesticide use behaviors were at a low level, with a tendency towards a high level of pesticide use behaviors. However, farmers had a moderate level of pesticide use behaviors at the ‘after pesticide application’ phase. This baseline study gives an insight into the range of pesticides used in the management of sweet corn pests and diseases in Thailand and found that 9 pesticides were used. The pesticides used in growing sweet corn have moderate toxicity. The major factors relating to pesticide use behaviors were the self-management and decision-making skills dimensions (p < 0.01). The lack of health literacy regarding pesticide calls for training for sweet corn farmers. To reduce the dependence on pesticides it is important to promote health literacy, especially in the cognitive skills, media skills and decision-making skills. The farmer has to find out what works best for them for reducing pesticide use when planting sweet corn. However, effective communication can lead to an improvement in pesticide use by farmers and may lead to improvements in environmental and ecological health.\n\n\nData availability\n\nOpen Science Framework: The association between health literacy and pesticide use behaviors among sweet corn farmers in the Pak Chong district of Thailand: a cross-sectional study. https://doi.org/10.17605/OSF.IO/KJ5M329\n\nThis project contains the following underlying data:\n\n- Data_Theerachai.csv (demographic and questionnaire response data)\n\n- Data Dictionary_Theerachari.csv (data dictionary for Data_Theerachai.csv)\n\n- Correct Responses_Theerachai.csv (scoring system for questionnaire responses in Data_Theerachai.csv)\n\nOpen Science Framework: The association between health literacy and pesticide use behaviors among sweet corn farmers in the Pak Chong district of Thailand: a cross-sectional study. https://doi.org/10.17605/OSF.IO/KJ5M3\n\nThis project contains the following extended data:\n\nQN-page1 - QN-page8 (questionnaire)\n\nData are available under the terms of access to the dataset requires registration, and is granted to those that wish to use the data for legitimate research purposes. A guide for how to apply for dataset access is available at: https://doi.org/10.17605/OSF.IO/KJ5M329\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThis research was supported by the Pakchong Nana Hospital of Public Health Ministry, Nakhon Ratchasima, Thailand. The authors wish to thank all of the sweet corn farmers that participated in this study.\n\n\nReferences\n\nTano ZJ: Ecological Effects of Pesticides. Pesticides in the modern world risks and benefits. Salaam, Tanzania, 2011. Publisher Full Text\n\nDamalas CA, Eleftherohorinos IG: Pesticide exposure, safety issues, and risk assessment indicators. Int J Environ Res Public Health. 2011; 8(5): 1402–1419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTawatsin A, Thavara U, Siriyasatien P: Pesticides used in Thailand and toxic effects to human health. Med Res Arch. 2015; 3: 1–10. Publisher Full Text\n\nChemLinked Team: Annual Report of Thailand Agricultural Pesticide Importation in 2014. China, 2015. Reference Source\n\nMinistry of Foreign Affairs: Sufficiency Economy Philosophy: Thailand’s Path towards Sustainable Development Goals. Thailand, 2017. Reference Source\n\nWorld Health Organization: The WHO recommended classification of pesticides by hazard and guidelines to classification. Stuttgart, Germany, 2009. Reference Source\n\nChan I, Devi NL: Pesticides Classification and its Impact on Human and Environment. Environ Sci Eng. 2017; 6: 140–158. Reference Source\n\nWorld Health Organization: Public Health effect impact of pesticide used in agriculture. England, 2019.\n\nTirado R, Englande AJ, Promakasikorn L, et al.: Use of agrochemicals in Thailand and its consequences for the environment. Greenpeace Research Laboratories Technical. Bangkok, Thailand, 2008. Reference Source\n\nAlewu B, Nosiri C: Pesticides and Human Health. Pesticides in the Modern World-Effects of Pesticides Exposure. University Campus STeP Ri Slavka Krautzeka, Rijeka, Croatia, 2011. Reference Source\n\nde Araujol JS, Delgadol IF, Paumgartten FJ: Glyphosate and adverse pregnancy outcomes, a systematic review of observational studies. BMC Public Health. 2016; 16: 472. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSorat W: The Relationship Between Health Beliefs, Pesticide Use and Safety Behaviors with Acute Poisoning Symptoms of Farmers in Chaiyaphum Province [thesis]. Bangkok: Mahidol University; 2004. Reference Source\n\nSuklim N, Raksanam B, Songthap A: Risk behaviors related agrochemical use among rubber farmers in Southern of Thailand (2014). European Journal of Research on Education. 2014; 2(2): 109–115. Publisher Full Text\n\nRatzan SC: Health literacy: communication for the public good. Health Promot Int. 2001; 16(2): 207–214. PubMed Abstract \| Publisher Full Text\n\nHepburn M: Health literacy, conceptual analysis for disease prevention. Int J Collab Res Intern Med Public Health. 2012; 4(3): 227–238. Reference Source\n\nBaker DW: The meaning and the measure of health literacy. J Gen Intern Med. 2006; 21(8): 878–883. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNutbeam D: Health literacy as a public health goal: a challenge for contemporary health education and communication strategies into the 21st century. Health Promot Int. 2000; 15(3): 259–267. Publisher Full Text\n\nNutbeam D: The evolving concept of health literacy. Soc Sci Med. 2008; 67(12): 2072–2078. PubMed Abstract \| Publisher Full Text\n\nNutbeam D: Defining, measuring and improving health literacy. HEP. 2015; 42(4): 450–455. Publisher Full Text\n\nAbbassy MM: Farmer’s Knowledge, Attitudes and Practices, and their Exposure to Pesticide Residues after Application on the Vegetable and Fruit Crops. Case Study: North of Delta, Egypt. J Environ Anal Toxicol. 2017; 7(5): 1–6. Publisher Full Text\n\nPanuwet P, Siripong W, Prapamontol T, et al.: Agricultural Pesticide Management in Thailand: Situation and Population Health Risk. Environ Sci Policy. 2012; 17: 72–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTawatsin A, Thavara U, Siriyasatien P: Pesticides used in Thailand and toxic effects to human health. Med Res Arch. 2015; 3: 1–10. Publisher Full Text\n\nOkonya JS, Kroschel J: A Cross-Sectional Study of Pesticide Use and Knowledge of Smallholder Potato Farmers in Uganda. Biomed Res Int. 2015; 2015: 759049. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRivera EP, Siriwong W, Taneepanichskul N, et al.: Health risk related to pesticide exposure in the agriculture system in Thailand: A systematic review. J Health Res. 2016; 30(Suppl 1): S71–S80. Publisher Full Text\n\nRijal JP, Regmi R, Ghimire R, et al.: Farmers’ knowledge of pesticide safety and pest management practices: A case study of vegetable growers in Chitwan, Nepal. Agriculture. 2018; 8(1): 16. Publisher Full Text\n\nOgg CL, Hygnstrom JR, Albert CA, et al.: Managing Pesticide Poisoning Risk and Understanding the Signs and Symptoms. NEBRASKA EXTENSION. 2006; 1–15. Reference Source\n\nLorenz AN, Prapamonto T, Narksen W, et al.: Pilot study of pesticide knowledge, attitudes, and practices among pregnant women in Northern Thailand. Int J Environ Res Public Health. 2012; 9(9): 3365–3383. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaksanam B, Taneepanichskul S, Siriwong W, et al.: Factors associated with pesticide risk behaviors among rice farmers in the rural community, Thailand. J Environ Earth Sci. 2012; 2(2): 32–39. Publisher Full Text\n\nPobhirun T: The association between health literacy and pesticide use behaviors among sweet corn farmers in the Pak Chong district of Thailand: a cross-sectional study. 2019. http://www.doi.org/10.17605/OSF.IO/KJ5M3" }	[ { "id": "47851", "date": "30 Apr 2019", "name": "Kishor Atreya", "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\nPage 1, Abstract:\n“89.98%” - 90%. What about the remaining participant? In the method section, the total participant mentioned was 161, and in results section it is said 90%. Confusion! “Functional literacy” - What is functional literacy? Briefly define the working definition of health literacy and pesticide use behavior in the abstract.\nPage 3, Introduction:\n“The top ten pesticides imported by Thailand…” - This is not required.\nPage 3, Methods:\n“194 sweet corn farmers were identified” - What was the sampling method? Random and purposeful? Any sample size calculations done? What was the population of the sweet corn farmer? “The research framework was approved…” - It is better suited in the text later - under the heading “ethical statement”. “who could read and write” - This study intentionally excluded illiterate sweet corn farmers? Why? These farmers might be at higher risk of exposure? “6 dimensions” - Any reference?\nPage 4, Methods:\n“36 items” - What are those 36 items? Please provide in the appendix. “low, moderate or high levels” - It is not clear that whether the authors demarcated these levels or derived from literature. “low, moderate and high standards of pesticide use behaviors” - Please provide 20 items in the appendix. “A score of <67 was considered to be low, a score of 67–69 was considered to be moderate and a score of >69 was considered to be high.” - Any literature published on these 3 categories? If so, please cite. “This analysis could also be performed using a non-proprietary software such as R.” - This is not required.\nPage 4, Results, The demographic characteristics of the 161 sweet corn farmers:\n“male” – Males. “had been” – Have been. “mixing” - What is the mixing method?\nPage 5, Results, Patterns of pesticide use:\n“four pesticide types” – Four types of pesticides. “pesticides” – Insecticides. “are” - Were\nPage 5, Results, Health literacy (HL) of pesticide use:\n“2. pesticide application, 3. pesticide application” – What is the difference between 2 and 3?\nPage 6, Discussion:\n“did not follow the current recommended dose or number of sprays” – I did not see any data on it in the result section. Ref 18 - This reference may be wrong here. Discussion is poor. Most of the discussion was done on the “consequences” of pesticide use, not on the results. The paper finds significant association between cognitive literacy and pesticide use behaviors, but not explained it in details. I would suggest having the results explained in the discussion section with some theory and recent literature.\nPage 7, Table 6:\nI see a number of zero values in the cells. How did authors consider these values in the Chi-square test?\nPage 8, Conclusions:\n“the self-management and decision-making skills dimensions (p < 0.01)” - This relationship should be explained in the discussion section. “The farmer has to find out…” - This is not required.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-448
https://f1000research.com/articles/8-260/v1	05 Mar 19	{ "type": "Research Article", "title": "Quantum of fluids in hospitalised patients with dengue fever and the presence of warning signs: a pilot cross-sectional study", "authors": [ "Ramayee Ramanathan", "Dheeraj Jain", "Stalin Viswanathan", "Ramayee Ramanathan", "Dheeraj Jain" ], "abstract": "Background: The World Health Organization in 2009 revised categories of dengue fever severity into classes A, B, and C based on the presence or absence of warning signs. The quantum of oral and intravenous fluids (IVF) in hospitalised patients with Group B (warning signs) have, to our knowledge, not been studied. Oral fluids in hospitalised patients and monitoring administration with the help of patients’ relatives have not been assessed. Methods: Consecutive patients with dengue fever and warning signs were studied for 48 hours after hospitalisation. Patients were asked to consume 4-5 l of fluids. Maintenance and bolus IVF were administered depending upon the presence of compensated or hypotensive shock. Intake and urinary output were monitored by the patient’s attendant. Total fluids (oral and intravenous) were divided by a number of anthropometric measurements. Significance of compensated and hypotensive shock, thrombocytopenia and 20% change in haematocrit, bleeding episodes and the need for transfusions, and organ impairment were considered in relation to the total amount of fluids received daily. Results: In total, 41 patients were studied. Patients with obesity and hypotensive shock received significantly more fluids on Day 1; taller patients and those with tachycardia, higher haematocrit and elevated creatine kinase correspondingly received more on Day 2. Hypotensive shock was significantly correlated with fluids/BSA and fluids/weight, while laboratory parameters correlated most strongly with fluids/BMI. Conclusions: Most adults with dengue and warning signs seem to require >100 ml/kg/day of fluids during their stay. Advising a similar amount of fluids at home during epidemics may further reduce the need for admissions. Participation of patients and their relatives in the bedside management of fluid administration may go a long way in preventing morbidity and mortality. Adults probably need a better anthropometric measurement to decide on the quantum of fluids to be given.", "keywords": [ "dengue", "fluid requirements", "warning signs", "guidelines", "Pondicherry outbreak" ], "content": "Introduction\n\nDengue fever is an acute arboviral febrile illness transmitted by Aedes mosquitoes and has four serotypes: DENV1, DENV2, DENV3, and DENV41. It has been reported in over 70 countries and is endemic in the tropics, where ≥2.5 billion people are at risk2–4. India is a category A endemic country2; in such countries dengue strains the already over-stretched health care system, and increases the economic burden due to hospitalization, absence from school, loss of productivity, loss of tourism revenue, and out-of-pocket expenses2,4. Even this burden is a gross underestimate due to underreporting of cases and deaths, poor networking of healthcare facilities, and interstate/inter-regional differences in the availability of optimal healthcare5. The number of reported dengue cases in Pondicherry, South India had fallen from >3500 in 2012 to <500 in the year 2016, with a reported total mortality of just six cases during those five years6.\n\nGuidelines by the World Health Organization (WHO) have been used to manage patients with dengue for ~45 years7. Until 2009, dengue cases were classified as dengue fever, dengue haemorrhagic fever and dengue shock syndrome, and appropriate management protocols were prescribed by WHO and national guidelines8. The revised classification by severity categorised patients into dengue fever without warning signs (Class A), with warning signs (Class B), and those with severe dengue (Class C)8. The warning signs include abdominal pain or tenderness, hepatomegaly, persistent vomiting, clinical fluid accumulation, mucosal bleed, lethargy and restlessness, and increase in haematocrit associated with fall in platelet counts. Patients in Group B must be admitted for the administration of fluids and observation during the critical phase, while patients in Group C should receive emergency care and/or urgent referral. Volume replacement guidelines by the WHO have been based on studies in children9. The quantum of fluids to be administered to patients in Group B have not been studied so far. Previously, only two studies had described the trends in fluid requirements over the patients’ entire hospital stay but using the earlier dengue severity classification10,11. Two other studies, one each from Malaysia and Nicaragua, had described the effectiveness of drinking one litre of fluid before hospital admission12,13.\n\nEven though only patients in class B and C require admission, most patients with dengue fever are often admitted due to inability to differentiate it from other acute febrile illnesses or from non-severe dengue itself9. Guideline-based administration of intravenous fluids (IVF) are based on patients’ weight and are generally a step-down regimen as the patient’s clinical and laboratory parameters improve. This is often not practical in primary care due to the shortage of doctors and nurses to monitor as prescribed in the guidelines, spacing constraints, and availability of 24-hour laboratory services. Surveillance for severe dengue is generally poor in countries with epidemics. This often leads to overcrowding at hospitals and clinics with dengue fever/mild illness. In association with overworked medical personnel and confusion among the lay public, this overcrowding often predisposes to less-than-optimal care for patients with severe dengue and increased mortality4.\n\nIn view of such a scenario, we decided to study patients hospitalized with dengue warning signs, wherein, they could be monitored and administered fluids, even with a low doctor/nurse-to-patient ratio, by informing patients in advance what their daily fluid intake was expected to be and using the assistance of their relatives in measuring fluid intake and output. This study aimed to find out the quantity of fluids adult patients with dengue fever and warning signs received to maintain hemodynamic stability and to discern which anthropometric measurement-based fluid requirements correlated best with manifestations of severe dengue.\n\n\nMethods\n\nThe study was conducted in Pondicherry during the 2017 dengue epidemic. All consecutive adolescent and adult patients admitted to Unit 3 of the Department of General Medicine, Indira Gandhi Medical College & Research Institute, with a confirmed diagnosis of dengue fever between 11 October 2017 and 08 November 2017 were recruited for the study. Ethics approval was obtained from the Institute Ethics Committee to obtain and record data from all febrile patients (IEC/PP/2016/44). Written informed consent was obtained from all participants to use their clinical and laboratory data.\n\nPatients with probable/confirmed dengue fever were admitted in the male medical ward (MMW), female medical ward (FMW), and a newly created dengue ward (DW). A total of 15 beds each had been added to the existing strength of FMW (60 beds) and MMW (70 beds) for ease of providing care. When these wards became full, newer patients were admitted into the DW (30 beds), which had a nurse available on all shifts, but without additional residents or house surgeons. The MMW and FMW were staffed by three nurses in the morning shift and two each in the afternoon and night shifts. The DW had two nurses in the morning and one each in the other two shifts. Each Unit was allotted 26 beds (26/130). Two faculty members, three residents, and three house surgeons of Unit 3 oversaw treatment of these patients. All doctors worked from 8:30 AM to 4:30 PM on weekdays, and from 8:30 AM to 1:00 PM on weekends. House surgeons managed night ward duties on a daily rotation, while residents stayed nights once a week. Patients could have one attendant/relative stay with them in the ward as part of hospital policy.\n\nAll patients 13 years or older examined in the outpatient or emergency department or with an acute febrile illness and a probable diagnosis of dengue fever were considered for the study. Such patients underwent a focussed clinical examination at the time of admission that included vitals, gums, skin, pallor, jugular venous pulse, oedema, hepatosplenomegaly, respiratory auscultation for crackles and diminished breath sounds. A complete blood count, liver function tests, renal function tests, creatine kinase, dengue IgM and NS1, urinalysis, chest radiograph, and electrocardiogram (ECG) were performed as part of routine care for febrile patients with suspected dengue. Patients were shifted to the wards after being stabilized in the emergency department. Those confirmed to have dengue fever by either dengue IgM or NS1 antigen positivity and had had warning signs at admission were considered eligible, and informed consent sought was obtained for inclusion in the study. Patients unwilling to participate, patients having dengue without warning signs, and those with an alternative diagnosis for the cause of acute febrile illness were excluded. Day 1 began at 8:00 AM on the following day after admission, and all patients were monitored for 48 hours until 8:00 AM of Day3.\n\nAll patients had been admitted into the hospital because of one or more of the following warning signs: persistent vomiting, abdominal pain, poor oral intake, dizziness, hypotension or shock, tachycardia during the afebrile phase of illness, severe thrombocytopenia, mucosal bleed, associated diabetes, pregnancy or alcoholism, and referral for admission from a peripheral health centre with poor transport facilities. Even though obese/overweight patients with difficult intravenous access are also considered as warning signs, we did not encounter difficulty in access and hence, they were not considered as a criterion for admission/warning sign8.\n\nPatients were classified into those with Class B dengue and those with Class C, based on WHO guidelines8. Class C included those with serositis, compensated and hypotensive shock, dysfunction in one or more organs, severe bleeding requiring blood products, severe thrombocytopenia, and a change in haematocrit by 20%. Postural symptoms were defined by the presence of either dizziness, light-headedness, feeling faint, palpitations, or sweating while sitting or standing from the supine position. Postural hypotension (PH) was considered when the systolic BP fell ≥20 mm and/or the diastolic blood pressure (DBP) fell ≥10 mm when the patient stood upright for two minutes after getting up from the supine position. In those patients with postural symptoms, blood pressure was recorded in the sitting position with legs dangling down. Postural BP measurements were done at least twice daily and the higher fall in BP was noted. The postural BP in the morning was taken under the supervision of the consultant taking rounds, while the evening and night shift recordings were taken by either the duty resident or house surgeon. Hypotension was considered when the systolic blood pressure was <100 mmHg. Tachycardia was when the pulse rate was ≥100 beats/minute. The presence of either tachycardia or postural hypotension defined compensated shock. One of the following was considered as hypotensive shock: pulse pressure was ≤20 mmHg, systolic blood pressure (SBP) was <90 mmHg, feeble pulse or the patient was restless. Serositis was defined by the new onset development of ascites and/ or pleural effusion. Organ impairment was defined by either/or the presence of acute kidney injury (Acute Kidney Injury Network criteria)14, transaminases elevation ≥3 times (hepatic dysfunction), CK elevation ≥3 times (myositis), and new onset cardiac arrhythmias. Patients were overweight when the body mass index (BMI) was ≥25 kg/m2. Bleeding was defined by the presence of new bleeding from any site- gums, nose (epistaxis), gastrointestinal (hematemesis, melena or haematochezia), respiratory(haemoptysis), uterine (menorrhagia/metrorrhagia) or skin (purpura or petechiae). Warning signs were defined by the presence of one or more of the following: abdominal pain, vomiting, dyspnea, dizziness, hepatomegaly, mucosal bleeding, diabetes mellitus, pregnancy, and alcoholism. A platelet count of <150x109/l was defined as thrombocytopenia, while severe thrombocytopenia was when the counts were <50×109/l. A significant change in haematocrit from Day 1 to Day 2 was considered when there was either a 20% increase or fall in haematocrit.\n\nAnthropometry (height, weight, BMI and body surface area) were measured on Day 1. BMI and body surface area (BSA) was calculated using the QxMD Calculate mobile app (version 7.0.6.0), using the De Bois formula. Pulse rate, SBP, DBP, pulse pressure, systolic postural fall (SBP fall) and diastolic postural fall (DBP fall) were measured at least twice daily during both days. More frequent measurements were done in those with postural symptoms and when receiving bolus IVF. Similarly, examination for abdominal tenderness, hepatomegaly, ascites, pleural effusion, altered consciousness were performed twice daily. Urea/creatinine, LFT and CK-total were done once at admission and again 48 hours later to monitor trends. CBC was performed twice daily during the 48 hours for all patients, and every 6th hourly in those with bleeding or 20% increase in haematocrit.\n\nAll patients were asked to drink 4–5 l of oral fluids cumulatively (a combination of water with or without oral rehydration salt, water with 5 tsp glucose, fresh juice, tender coconut water, buttermilk, milk, and dilute millet or rice porridge). The patients and their relatives were instructed to keep five 1-l bottles at the bedside to be filled up with water or oral rehydration salts each morning for easy monitoring. Other beverages were accounted as follows using two disposable paper cups of 200 ml and 100 ml each: a glass of juice, water with 5 tsp glucose, tender coconut water or porridge as 200 ml; 1 glass of buttermilk or milk as 100 ml. The total quantity of oral fluids was rounded-off to the nearest 100 ml. IVF was initiated for those in with postural symptoms, hypotension, shock, abdominal pain, persistent vomiting, or those with poor oral intake. Either normal saline or ringer lactate was administered as maintenance fluids, at the rate of 100 ml/hour, calculated using the following formula: {1500 ml+[weight (kg) × 20 ml/kg/day]=2500 ml, for a 50-kg person}. Boluses of normal saline (500 ml) were given for those patients in shock and in those with significant postural fall in blood pressure. Dextrose saline (5% DNS) was administered for patients with poor calorie intake and in those with hepatic dysfunction. The empty fluid bottles were retained at the patient’s bedside cupboard to keep count of IVF to the nearest 500 ml. The attendants of patients who could document both oral and IVF intake on paper were exempted from keeping bottle counts. Fluid intake was measured at the end of Day 1 and Day 2. Urine output was measured every 6th hourly. The total fluid counts (oral +intravenous) for each day was divided by each patient’s weight, height, BMI and BSA. A measuring jar cut out from an IVF bottle, was used for measuring urine output (Figure 1). Attendants were instructed to inform and/or note down the colour and quantity of urine and urgently bring to notice if there was no urine output for six continuous hours. Outcome measures were the total oral fluids and total intravenous fluid administered, low urine output (<500 ml/day), bleeding episodes and need for transfusion, compensated and hypotensive shock, and organ dysfunction such as hepatitis and myositis.\n\nData were entered in an Excel spreadsheet and thence imported into IBM SPSS for Windows v22 for statistical analysis. Frequencies of clinical symptoms, examination findings, and severe organ involvement were calculated. Chi-square analysis (Fischer’s exact test for frequencies <5) was used to compare the presence of symptoms, signs, and investigations among those who had received more than 5 l fluid/day with those who had not. Means ± standard deviations were calculated for continuous variables (clinical, lab, and treatment) and compared between the two groups receiving and not receiving 5 l fluids on both days using t-test. Bivariate correlations were performed between fluid administration (based on weight, height, BMI, and BSA on both days) and clinical/lab values. P<0.05 was considered to indicate a statistically significant difference.\n\n\nResults\n\nOf the 43 patients, 30 were female. Most patients were admitted on the fourth (n=9), fifth (n=8), and seventh (n=7) days of fever. Males had a significantly higher postural fall compared to females and had higher haematocrit and CK values. At admission, all patients had had warning signs, while 37 continued to have warning signs on Day 1, and on further evaluation and investigations, 33 of them also had signs of severe dengue (Class C) (Table 1). In total, 15 had serositis and/or organ involvement in the form of either hepatitis, AKI, myositis, or cardiac dysfunction (1 case of Brugada Type 1 ECG pattern). In total, 27 were afebrile on Day 1, and 36 on Day 2. BMI was comparable between males and females (24.79±6.45 versus 23.98±4.92). Thrombocytopenia was seen in 12 and 11 patients on Day 1 and Day 2, respectively, with bleeding manifestation in seven (three with gum bleeds and one each with epistaxis, metrorrhagia, haemoptysis and hematochezia). Only four patients had an increase in haematocrit ≥20% on Day 2 compared with the previous day. Obese individuals and patients with hypotensive shock received significantly more fluids on Day 1 than adolescents (Table 2). There was no significant difference in fluids received in those with and without warning signs. Bleeding was not significantly related to platelet counts, haematocrit, liver dysfunction, or CK elevation. Serositis, hepatitis, severe thrombocytopenia, bleeding, and compensated shock did not correlate with any anthropometry-related fluid calculation. Hypotensive shock had significant correlations with fluids/BSA and fluids/weight, while laboratory parameters correlated with best with fluids/BMI (Table 3). None of them developed fluid overload, pulmonary oedema, ARDS or admission into intensive care unit (ICU). There was no mortality. Raw data are available on Harvard Dataverse15.\n\nValues are given as mean ± standard deviation where indicated.\n\nECG, electrocardiogram; HR, heart rate; BMI, body mass index; BSA, body surface area; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase; CK, creatine kinase.\n\nValues are given as mean ± standard deviation.\n\nBSA, body surface area;SGOT, serum glutamic oxaloacetic transaminase; CK, creatine kinase.\n\nPatients’ inability/hesitancy to drink fluids as instructed by the staff were due to poor taste, nausea, vomiting, abdominal pain, dizziness on sitting up or walking to the restroom, inability to use the restroom following increased fluid intake, and poor cleanliness of the common restrooms. Patients referred from peripheral health centres for admission also felt that care was incomplete without IVF, preferring IVF over oral fluids, and often demanded IVF. In those who received IVF, problems encountered include wrong beliefs that they should not eat or drink while receiving IVF, unavailability of IVF stands on occasions (due to requirement by most patients in the ward), thrombophlebitis, delay in nurse or a doctor reaching them to disconnect the IV line so that they could use the toilet and then reconnect it again, difficulty in sleeping in a comfortable position because of the cannula, and washing after using the toilet (IV cannula in the left wrist/hand).\n\n\nDiscussion\n\nThere are 50–100 million new cases of dengue annually, with the vast majority of them in individuals >15 years of age4,16. Dengue began being reported in Pondicherry from 2007 onwards1. A study from a hospital in Pondicherry during 2017 described the peak incidence of dengue between September and October; the dengue seroprevalence among antenatal mothers and children with fever was found to be 25.8%17. Severe dengue is more common in children18. The mean age of patients with severe dengue in our study was 30±12 years, compared to 26±11 years in those without severe dengue. Reports from India show that confirmed cases are being described mostly in the 21–30 years age group1. Even though dengue shock requires admittance to an ICU, we managed all patients in the wards due to a shortage of beds in the ICU3. None of our patients developed other indications of ICU care such as ARDS, cardiogenic shock, encephalitis, or secondary pneumonia.\n\nFluid calculation during the critical phase is calculates as follows: 100 ml/kg for the first 10 kg (1000 ml), 50 mL/kg for the second 10 kg(500 ml) and 20 ml/kg for >20 kg up to 50 kg (600 ml), and 5% deficit, calculated as 50 ml/kg up to 50 kg (2500 ml) which yields a total of 4600 ml for a 50 kg person for about 48 hours19. Since 33/37 patients had severe dengue, the fluids administered were much higher than this amount during each day of the two-day study. Some studies have shown that the new WHO guidelines increased the sensitivity of identifying patients with severe illness, with a risk of over-admission and increased workload, which would not be desirable in endemic regions that were resource-limited7. In a study from a tertiary hospital at Singapore, some cases of dengue with warning signs were safely managed in outpatient settings, but this practice cannot be extrapolated to other settings of primary/secondary care7. Most patients with severe illness progressed within a day of developing warning signs7. Thus, our study attempted to find out whether a range of fluid requirement would satisfy most patients (at least in a hospital) with dengue and warning signs without the minute assistance of a doctor or a nurse.\n\nOnly four studies have quantified the fluid requirements of patients with dengue, among which two have been performed in hospitalised patients10–13. Three had used the earlier WHO classification for severity of dengue: dengue fever, dengue haemorrhagic fever and dengue shock syndrome. The actual requirements of fluids in these three subtypes have not been quantified and vary with individuals; it depends on the availability of resources, staff, and place of treatment. Two studies had reported fluid requirements from admission to discharge10,11. A large prospective study of 1501 patients in Asia and Latin America with children constituting 65% described fluid accumulation, shock, and respiratory distress in relation to IV fluids, especially boluses. Median IVF volumes for maintenance, rehydration, and resuscitation were 50, 50 and 64.7 l/kg, respectively. The quantity of oral fluids consumed was not reported11. Another Sri Lankan study had shown that the quantity of fluid consumed had no impact on the development of third space fluid accumulation9. We are the first to assess fluid (oral and IVF) requirements based on the severity classification of A, B, and C. We had practical difficulties with both oral and IVF (as listed under Results), but persisted in explaining the need for fluids, especially by mouth.\n\nFluids losses into the third spaces may occur even prior to the fourth day of fever and it is imperative that fluid replacement begins early, especially prior to admission, beginning at home10. Our study showed that the maximum total fluids (13,700 and 14,100 ml on Day 1 and Day 2, respectively) had been administered on the fourth day of fever (Figure 2). During the first two days of fever, it is difficult to predict whether the disease may progress to shock (compensated or hypotensive) and hence fluid supplementation is important. In the Sri Lankan study auditing the trends in fluid requirements, 6267 ml was the mean requirement on the fourth day of fever similar to that of our study (6141 ml on Day 1 and 6433 ml on Day 2)10.\n\nThe Malaysian study showed that reading out advice from a dengue home care card to patients attending primary care clinics improved fluid intake by at least one litre and reduced hospitalization rates12. They also found that a fluid chart maintained by the patient improved awareness of fluid intake and enhanced compliance. In Nicaragua, where the amount of fluid was also quantified, drinking five glasses of fluids prior to being seen by a doctor was protective against hospitalization13. The various liquids ingested in that study included juices, lemonade, milk, coffee, tea, and oral rehydration salts. We did not advise tea or coffee because of their weak diuretic action. We also suggested tender coconut water, dilute millet or rice porridge. Promoting high fluid intake was suggested to make an economic impact in countries with dengue fever13. We suggested an empirical 4–5 litre count based on the 100 ml/kg for an average 50 kg person, to account for both maintenance and replacement fluids arising from losses due to vomiting, diarrhoea, high fever, anorexia and poor intake in hot and humid weather conditions.\n\nVolume replacement guidelines are based on studies in children and hence caution was advised in order to avoid fluid overload16. Intravenous fluids were given at a mean volume of 110 ml/kg over a mean of 25.3 hours in infants in a Vietnamese study which studied both patients with dengue hemorrhagic fever and dengue shock syndrome16. This contrasted with a mean IVF volume of 40.44 ml/kg (2318.6 ml/57.33 kg) on Day 1 and 29.69 ml/kg (1702.33 ml/57.33 kg) on Day 2 in our study. Hence the requirement of administered fluids is much lower when considered for their weights. A different anthropometric measurement may perhaps be useful in adults to gauge actual requirements, since the BSA and BMI correlated better with laboratory parameters of severe dengue in our study. Fluids/BSA correlated best with hypotensive shock on both days, while fluids/BMI correlated significantly with all the lab predictors of severe dengue.\n\n\nLimitations\n\nThis study had very small numbers due to the fact it was an audit of treatment practices in a single treating unit in Medicine (total of five units) during an epidemic that lasted 2 months. The dengue species was not known. The blood pressure was measured using a mercury sphygmomanometer, where errors in a few millimetres could probably misclassify patients with compensated and hypotensive shock. Also, some patients with severe postural symptoms had their BP checked in the sitting position, which could have led to a higher pulse pressure being recorded. The ECG heart rate was documented only at admission and patients could have been misclassified as having or not having a warning sign during Day 1 of the study. For logistic reasons, the study began only at 0800 AM of the following day after admission and hence the fluid administration for patients with shock or warning signs during the previous hours was not quantified. This amount may have been higher since all patients had been admitted initially with warning signs.\n\n\nConclusions\n\nMost adults hospitalised with dengue fever and warning signs seem to require >100 mL/kg/day of fluids during their hospital stay without developing symptoms of fluid overload. This is over and above what has been described in the national guidelines, which are weight-based calculations. Abdominal pain, overweight individuals, tachycardia on ECG, hypotensive shock, increasing haematocrit and elevated CK levels were significantly associated with having received >5 l/day. Since some patients developed warning signs on the third day of fever, it would be prudent to sometimes lower the threshold for admission in patients who come to the emergency department. Advising a larger amount of fluids (as in our study) at home during dengue epidemics could probably further reduce the need for admissions and/or development of severe dengue- a fraction of the fluid quantity that we administered our patients had already shown to reduce hospitalization rates in two other studies. In resource-poor settings, letting patients and their attendants participate in the acute bedside management of dengue with warning signs may go a long way in preventing morbidity in dengue fever.\n\n\nData availability\n\nHarvard Dataverse: Quantum of fluids in hospitalised patients with dengue and warning signs- a pilot cross-sectional study. https://doi.org/10.7910/DVN/SKEJAM15.\n\nThis project contains information on the fluids consumed and patient blood values.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nChakravarti A, Arora R, Luxemburger C: Fifty years of dengue in India. Trans R Soc Trop Med Hyg. 2012; 106(5): 273–82. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization, Regional Office for South East Asia: Comprehensive guidelines for prevention and control of dengue and dengue hemorrhagic fever: revised and expanded edition. New Delhi, India: World Health Organization; 2011. Reference Source\n\nTeixeira MG, Barreto ML: Diagnosis and management of dengue. BMJ. 2009; 339: b4338. PubMed Abstract \| Publisher Full Text\n\nGubler DJ: Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol. 2002; 10(2): 100–3. PubMed Abstract \| Publisher Full Text\n\nGarg P, Nagpal J, Khairnar P, et al.: Economic burden of dengue infections in India. Trans R Soc Trop Med Hyg. 2008; 102(6): 570–7. PubMed Abstract \| Publisher Full Text\n\nSahanaa C, Mishra AK, Bazroy J: Trend of morbidity and mortality of dengue in Tamil Nadu and Puducherry, South India. Int J Community Med Public Health. 2018; 5(1): 322–5. Publisher Full Text\n\nLeo YS, Gan VC, Ng EL, et al.: Utility of warning signs in guiding admission and predicting severe disease in adult dengue. BMC Infect Dis. 2013; 13: 498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization and the Special Programme for Research and Training in Tropical Diseases: Dengue Guidelines for diagnosis, treatment, prevention and control: New edition. France; World Health Organization; 2009. PubMed Abstract\n\nPremaratna R, Ragupathy A, Miththinda JK, et al.: Timing, predictors, and progress of third space fluid accumulation during preliminary phase fluid resuscitation in adult patients with dengue. Int J Infect Dis. 2013; 17(7): e505–e509. PubMed Abstract \| Publisher Full Text\n\nKularatne SA, Weerakoon KG, Munasinghe R, et al.: Trends of fluid requirement in dengue fever and dengue haemorrhagic fever: a single centre experience in Sri Lanka. BMC Res Notes. 2015; 8: 130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosenberger KD, Lum L, Alexander N, et al.: Vascular leakage in dengue--clinical spectrum and influence of parenteral fluid therapy. Trop Med Int Health. 2016; 21(3): 445–53. PubMed Abstract \| Publisher Full Text\n\nNasir NH, Mohamad M, Lum LCS, et al.: Effectiveness of a fluid chart in outpatient management of suspected dengue fever: A pilot study. PLoS One. 2017; 12(10): e0183544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarris E, Pérez L, Phares CR, et al.: Fluid intake and decreased risk for hospitalization for dengue fever, Nicaragua. Emerg Infect Dis. 2003; 9(8): 1003–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopes JA, Jorge S: The RIFLE and AKIN classifications for acute kidney injury: a critical and comprehensive review. Clin Kidney J. 2013; 6(1): 8–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nViswanathan S: Quantum of fluids in hospitalised patients with dengue and warning signs- a pilot cross-sectional study. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/SKEJAM\n\nNguyen TH, Nguyen TL, Lei HY, et al.: Volume replacement in infants with dengue hemorrhagic fever/dengue shock syndrome. Am J Trop Med Hyg. 2006; 74(4): 684−691. PubMed Abstract \| Publisher Full Text\n\nGopal V, Aruna, Sujatha P: Seroprevalence of Dengue in Antenatal and Paediatric Patients - In a Tertiary Care Hospital, Puducherry. Int J Curr Microbiol App Sci. 2018; 7(6): 667–72. Publisher Full Text\n\nDung NM, Day NP, Tam DT, et al.: Fluid replacement in dengue shock syndrome: a randomized, double-blind comparison of four intravenous-fluid regimens. Clin Infect Dis. 1999; 29(4): 787–94. PubMed Abstract \| Publisher Full Text\n\nMinistry of Health, Sri Lanka- National Guidelines, in collaboration with Ceylon College of Physicians: Guidelines on management of dengue fever and dengue haemorrhagic fever in adults. Colombo, Sri Lanka: 2010. Reference Source" }	[ { "id": "45296", "date": "25 Mar 2019", "name": "Siripen Kalayanarooj", "expertise": [ "Reviewer Expertise dengue" ], "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 underscored warning signs (WS) but the definition they used was different from the references1 that they cited. Because of the different WS definitions, the enrolment criteria and exclusion criteria were not clear, which resulted in a non-homogeneous study population. They include both shock and non-shock patients, i.e. dengue with WS and severe dengue on the time of admission. So, the results might not be relevant. In addition, in Table 1 the results revealed that on day 1, the number of cases with compensated shock increased from 4 (day 1) to 7 (day 2) in the <5L group and reduced from 7 (day 1) to 6 (day 2) in the ≥5L group. The number of cases with hypotensive shock reduced from 7 (day 1) to 5 (day 2). Were the shock patients on day 1 and 2 the same patients or different ones?\nThe same results on day 2: the number of cases with compensated shock increased from 3 (day 1) to 6 (day 2) and number of cases with hypotensive shock increased from 1 to 2 and in the <5L group. In the ≥5L group, compensated shock reduced from 8 (day 1) to 7 (day 2) and from 6 (day 1) to 3 (day 2) for hypotensive shock. Were the shock patients on day 1 and 2 the same patients or different ones? For the above results, even in the ≥5L group, the amount of fluid didn't seem very effective in maintaining hemodynamic stability. Still, there were more than 50% of cases with shock, or that developed shock (if not the same patients).\nPlease clarify number of study cases, 41 (in the abstract) or 43 (in the results and Table 1). Because of different definitions, the number of patients with severe dengue increased, but by looking at the mean platelet count, mean HCT and mean AST/ALT, it seems that the majority of enrolled case were not severe. But in the more severe cases, this fluid amount could not prevent shock in at least 14 cases (33%) in this study.\n\nThe following definitions in this study were different from standard:\nWarning signs (WS) that were not in the references: poor oral intake, dizziness, hypotension or shock, tachycardia during the afebrile phase, severe thrombocytopenia, associated diabetes, pregnancy or alcoholism, referral for admission from a peripheral health center and obese patients. Hypotension was considered when systolic blood pressure was <100 mmHg. Tachycardia defined compensated shock. Hypotensive shock = pulse pressure 20 mmHg or systolic BP <90 mmHg or feeble pulse or the patients was restless. Organ impairment was defined as:\nTransaminase elevation >3 times. CK elevation >3 times.\n\nThrombocytopenia = platelet count, 150,000/cumm.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4567", "date": "15 May 2019", "name": "Stalin Viswanathan", "role": "Author Response", "response": "Dear Sir,The following modifications have been made by us with regard to your comments for your perusal. The abstract has been corrected to include 43 patients as in the rest of the manuscript. The definitions of warning signs have been included as in WHO 2009 guidelines. The other criteria such as pregnancy and diabetes which are only indicators for admissions and categorizing under Group B have been mentioned separately. nevertheless, both the male diabetic and pregnant lady had warning signs at admission. Tachycardia as a single criterion for compensated shock has been removed. Hypotension has been rewritten as BP <90mmHg and the results have been modified accordingly. Organ impairment with regard to hepatitis has been removed. Since myositis is only part of the expanded dengue syndromes without a clear cut-off level, 3x levels were originally considered significant. But they have been excluded now. Only transaminases and CK levels are mentioned. Thrombocytopenia has been removed and only severe thrombocytopenia <50,000 has been retained. As you have rightly remarked, the majority was not severe. Even the severe cases, were because of having a pulse pressure <20mmHg, postural drop in BP or hypotension. Patients were taken into the study when the admitting diagnosis was warning signs - they could have had postural hypotension, or they may have worsened further when the study began at 08 AM. We did not foresee this, and the true picture of having some severe dengue among the cohort was available only after the statistical analysis. In view of alteration in definitions, the numbers of hypotensive and compensated shock have changed. These have been incorporated, along with the clarification in the results regarding new patients who developed compensated or hypotensive shock. A new row (new compensated shock on Day 2) has been added in the table. Regards,Stalin V" } ] }, { "id": "45298", "date": "02 Apr 2019", "name": "Anon Srikiatkhachorn", "expertise": [ "Reviewer Expertise Dengue clinical manifestations", "disease mechanisms", "and clinical management." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Ramathan and colleague describes the fluid administration in patients hospitalized for dengue with warning signs (DWS) and evaluate the characteristics related to the amount of fluid received. The important aspect of this work in the “real world” setting where close monitoring of all dengue cases by medical staff may be less than optimum and the patients and relatives play important roles in the primary intervention, namely fluid administration.\nAlthough the manuscript addresses an important issue, there are a number of problems in the methodologies and interpretations. The number of cases included in this report is rather small and the enrolment criteria included those that are not true warning signs. These include co-morbidity such as pregnancy, alcoholism, and social reasons. It is not clear how many of cases were enrolled based on these criteria. Further, definitions of severe dengue in this manuscript are different to those according to the WHO 2009 classification and have been expanded to include: a 20% increase in hematocrit reading, at least 3 fold increase in transaminase or CPK levels. The expanded definitions for warning signs and severe dengue in this manuscript may cause confusions to readers and limit the generalizability of the findings in this report.\nAlthough detailed information regarding staffing of the medical wards was provided this does not clearly convey the measurable indicators of work load and man power utilized for patient care. Measurable indicators such as the ratio of cases to medical and nursing staffs should provide a clearer picture.\n\nTachycardia was used as an indicator of compensated shock. Tachycardia itself can occur from other reasons including fever, anxiety and discomfort that are not related to volume status. It is not clear whether all cases with tachycardia had been tested for postural hypotension before assigning them as severe.\n\nIt is notable that the average amounts of fluid received by dengue cases in this study were substantially more than previously reported. A manuscript from Kalaratne et aI1 was referenced to indicate fluid requirement comparable to this study. Upon reviewing this reference, the amount of fluid administered in DF and DHF cases was approximately 3000 ml per day, which is significantly less than the amount given in this current work. Although there were no report of respiratory complications such as pulmonary edema in this series, careful administration of fluid must be exercised to avoid fluid overload and respiratory complications. This also calls into question the accuracy of the estimated amount of fluid in this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "4568", "date": "15 May 2019", "name": "Stalin Viswanathan", "role": "Author Response", "response": "Dear Sir,The following modifications have been made with regard to your comments for perusal. As you rightly said, this is a small study and it has been mentioned in the title - a pilot study, partly because of the short duration of the epidemic and a single centre study. All patients were enrolled because of the admitting diagnosis - \"Dengue with warning signs\". It is only after complete statistical analysis, that some of them have been found to have other features of severe dengue such as postural hypotension or an arrhythmia discovered on ECG The enrolment criteria did not include pregnancy, obesity or alcoholism or diabetes as stated. These factors were considered by the admitting emergency department physician. But the diabetic male and pregnant lady in our study had warning signs. The statement in the manuscript as \"warning signs\" have been suitably modified. The expanded definitions to include sever dengue had been removed and the original retained. Thus the results section and the tables have been corrected accordingly. Thus hepatitis, myositis and hematocrit increase have been deleted. The nurse-patient and doctor-patient ratio have been added in the section accordingly. As per your comments, tachycardia alone without other symptoms have been NOT considered as compensated shock. Thus the tables with compensated shock and hypotensive shock have been changed. All patients (not only with tachycardia) were checked for postural hypotension on both days. Our patients received more fluids than the one cited in the text, partly because some of them had compensated shock or hypotensive shock along with one or more warning signs noted at the time of admission. Thus they received more boluses and instructions to attendants to serve more fluid drinks. Patients with warning signs are often not checked for postural fall in the emergency department, and similarly, the pulse pressure is often not keenly looked at, and narrowing pulse pressure is often missed. Only in one 30-year-old lady with metrorrhagia, dyspnea, and compensated shock was there a suspicion of fluid overload since she received 10200mL on Day 1and hence only 1750mL on Day2. But CXR and USG showed no evidence of either effusion or edema and, therefore she received only RBC transfusions for uterine bleeding. Regards and thank you.Stalin V" } ] } ]	1	https://f1000research.com/articles/8-260
https://f1000research.com/articles/8-659/v1	14 May 19	{ "type": "Research Article", "title": "Does handwriting the name of a potential trial participant on an invitation letter improve recruitment rates? A randomised controlled study within a trial", "authors": [ "Jennifer McCaffery", "Alex S. Mitchell", "Caroline Fairhurst", "Sarah Cockayne", "Sara Rodgers", "Clare Relton", "David J. Torgerson", "OTIS Study Team", "Alex S. Mitchell", "Caroline Fairhurst", "Sarah Cockayne", "Sara Rodgers", "Clare Relton", "David J. Torgerson" ], "abstract": "Background: Randomised controlled trials (RCTs) often fail to recruit to target, resulting in a lack of generalisability of findings. A wide range of strategies for potentially increasing recruitment have been identified; however, their effectiveness has not been established. The aim of this study within a trial (SWAT) was to evaluate the effectiveness of handwritten personalisation of an invitation letter as part of a trial recruitment pack on recruitment to a host RCT. Methods: A pragmatic, two-armed RCT was conducted, embedded within an existing falls prevention trial (OTIS) in men and women aged 65 years and over living in the community. Participants were randomised 1:1 to receive an OTIS recruitment pack containing an invitation letter on which their name was handwritten (intervention group), or one on which it was printed (control group). The primary outcome was randomisation into the host trial. Secondary outcomes related to trial eligibility and retention. Analyses were via logistic regression and Cox Proportional Hazards regression. Results: Of the 317 SWAT participants, 12 (3.8%) were randomised into the OTIS trial: 3 (handwritten: 3/159 [1.9%]; printed: 9/158 [5.7%]; difference -3.8%, 95% CI -8.0% to 0.4%). There was weak evidence, against the intervention, of a difference in the likelihood of participants being randomised into the host trial between the two groups (OR 0.32, 95% CI 0.08 to 1.20, p=0.09). There were no statistically significant differences between the intervention and control groups on any of the secondary outcomes. Conclusions: There was no evidence that personalisation of invitation letters improved recruitment to the OTIS trial. However, due to the small sample size, the results should be interpreted with caution. These findings need to be replicated across larger studies and wider populations. Registration: ISRCTN22202133.", "keywords": [ "Recruitment", "Randomised Controlled Trial", "Embedded trial", "Personalisation", "Handwritten" ], "content": "Introduction\n\nRandomised controlled trials (RCTs) are regarded as the gold standard design to evaluate the effectiveness of interventions in health research1–3. However, a recent review of RCTs funded by the National Institute of Health Research (NIHR) in the UK found that only 56% of RCTs reviewed achieved their planned sample size4.\n\nTrialists are well aware of recruitment challenges and have adopted a wide range of strategies to achieve and retain their sample size, including gifts, reminders and enhanced cover letters5,6. A recent Cochrane review has evaluated strategies used to improve recruitment to RCTs7. This review identified 68 trials involving more than 74,000 participants. The key conclusions from this review were that there was high-certainty evidence for three methods. The first is that informing participants what they will receive in the trial improves recruitment. The second is that phoning people who do not respond to postal invitations can be effective. Finally, using a tailored, user-tested information sheet makes little or no difference to recruitment. The review found that of the 72 strategies evaluated, only seven involved more than one study; therefore, additional studies are needed to evaluate the effectiveness of strategies to improve recruitment.\n\nOne potential strategy that has been found to be effective at improving the responses to postal questionnaires8 is making trial documentation more personal. In a review by Edwards et al.8, data from 58 trials demonstrated that the odds of returning a questionnaire was increased by more than one tenth (OR 1.14, 95% CI 1.07 to 1.22) when questionnaire material was made more personal. However, there was a wide range of ‘personalisation’ investigated within these trials (e.g. hand-addressing envelopes, signing letters personally etc.). Another systematic review looked specifically at the effect of personally addressed and/or hand-signed letters on questionnaire response. This review found that there was a positive effect on response rates when letters were personally addressed, which increased when letters were also hand-signed9.\n\nOur aim was to undertake a study within a trial (SWAT) to determine whether the number of participants recruited to a trial can be improved by writing the potential participant’s name by hand, versus printing the name, on the invitation letter. By embedding a study within a RCT currently being coordinated by the York Trials Unit (YTU; University of York), the NIHR-funded OTIS study10, the effects of the intervention could be tested within a pragmatic context without the additional costs of participant recruitment.\n\n\nMethods\n\nThis trial was embedded within the NIHR Health Technology Assessment (HTA) funded Occupational Therapist Intervention Study (OTIS)10 (Programme grant number 14/49/149). The OTIS study is an RCT that aims to evaluate the clinical and cost effectiveness of an occupational therapist (OT) intervention to reduce falls in high risk older people. Ethical approval for the OTIS trial and this embedded study was given by the West of Scotland Research Ethics Service (WoSRES); Health Research Authority approval and the Department of Health Sciences Research Governance Committee at the University of York. This study within a trial was registered with the ISRCTN registry as part of the host trial registration (ISRCTN22202133; date registered: 20th June 2016)\n\nThe OTIS trial is a modified cohort11, pragmatic, multicentre, two-armed RCT. Detailed methods of the main trial have been published elsewhere10. Participants for OTIS were recruited by mail out of recruitment packs to: i) participants in the Yorkshire Health Study (YHS)12; ii) cohorts of previous trials held by the YTU13–15; iii) potentially eligible patients identified in GP practice databases; and iv) via opportunistic screening. This embedded trial involved potential participants who were approached in the first mail out from the YHS (see Figure 1 for a participant flow diagram). Recruitment to the embedded trial began on the 20th of April 2017 and follow-up ended on the 22nd August 2018.\n\nAfter receiving a recruitment pack (consisting of an invitation letter, participant information sheet, consent form, contact form and screening questionnaire) participants were asked to return their completed screening questionnaire and consent form to researchers based at the YTU if they wished to take part in the study. Participants were eligible to take part in the OTIS trial if they were aged 65 years or over, lived in the community, had fallen in the past 12 months or had a fear of falling, and were willing to receive a home visit from an OT. Participants were ineligible if they were unable to walk 10 feet (even with a walking aid), had dementia, lived in a residential or nursing home, had poor levels of English (with no access to assistance), had received an OT assessment for falls prevention in the last 12 months or were on a waiting list for an assessment, or had not returned a completed falls calendar. Participants who were eligible except for the fact that they had not fallen in the last 12 months and did not report a fear of falling were rescreened at intervals until they asked not to continue participation or they became eligible. Once eligible, participants were sent a baseline questionnaire and pack of falls calendars. Participants who completed their baseline questionnaire and falls calendars then became eligible for randomisation into the OTIS trial. For the main OTIS trial, participants were randomised to an environmental assessment by an OT or to the control group.\n\n\nRandomisation and blinding\n\nThis trial was embedded in the OTIS trial, and included potential OTIS participants sent a recruitment pack as part of the first mail out to consenting members of the YHS. Recruitment packs were assigned a unique identification number. Block randomisation was used to allocate the recruitment packs in a 1:1 ratio to either the control group or the intervention group, using one large block the size of the mail out (n=317). Generation of the allocation sequence was undertaken by the OTIS trial statistician, who was not involved with the production of the recruitment packs, using Stata version 13.\n\nThe control group received the standard recruitment pack with an invitation letter with their name printed in the salutation. The letter was printed on one page of A4 and is shown in Extended data, Supplementary File 116.\n\nThe intervention group received the standard recruitment pack with an invitation letter with their name handwritten in the salutation. The names were written by a researcher in in the form Mr/Ms Firstname Surname. The letter was printed on one page of A4 and is shown in Extended data, Supplementary File 216.\n\nThe primary outcome was the proportion of participants included in the embedded trial, who went on to be randomised to the host OTIS trial.\n\nThe secondary outcomes were:\n\nproportion of participants who returned a screening form\n\ntime to return screening form\n\nproportion of participants who fulfilled the eligibility criteria on their initial screening form apart from the criterion relating to falls within past 12 months or fear of falling\n\nproportion of participant who were eligible on their initial screening form\n\nproportion of participants who remained in the trial at three months post randomisation (defined as returning at least the first three months’ worth of falls calendars from the date of randomisation).\n\nWe randomised 317 participants who were due to be mailed out a recruitment pack about the OTIS trial by the YHS. This sample size is sufficient to detect a 10% absolute difference in the percentage of participants who go on to be randomised (from 10 to 20%) between the two groups at 80% power and a two-sided alpha level of 0.1.\n\nData were analysed on the basis of intention-to-treat and all hypothesis tests were two-sided at the 10% significance level. Categorical data were compared using logistic regression models and time to response data using a Cox proportional hazards model. An additional logistic regression model used to analyse whether participants remained in the trial 3 months post-randomisation was adjusted for the OTIS main trial group allocation (usual care or intervention). The odds ratio (OR) or hazard ratio (HR) from each model associated with the embedded trial allocation is presented along with the corresponding 95% confidence interval (CI) and p-value. All analyses were conducted using Stata version 15.\n\nA completed CONSORT checklist is available at Open Science framework16.\n\n\nResults\n\nRandomised to OTIS main trial. Of the 317 embedded trial participants, 12 (3.8%) were randomised into the OTIS trial (handwritten: 3/159 [1.9%]; printed: 9/158 [5.7%]; difference -3.8%, 95% CI -8.0% to 0.4%). There was weak evidence of a difference in the likelihood of embedded trial participants being randomised into OTIS between the two groups (OR 0.32, 95% CI 0.08 to 1.20, p=0.09), in favour of the control group.\n\nReturned screening form. In total, 49 (15.5%) of the 317 embedded trial participants returned a screening form (handwritten: 22/159 [13.8%]; printed: 27/158 [17.1%]; difference -3.3%, 95% CI -11.2% to 4.7%). There was no evidence of a difference in the likelihood of embedded trial participants returning a screening form between the two groups (OR 0.78, 95% CI 0.42 to 1.44, p=0.42).\n\nTime to return of screening form. For the screening forms returned, the median time to return was 26 days (interquartile range [IQR] 20 to 60) in the handwritten arm and 26 days (IQR 20 to 204 days) in the printed arm. There was no evidence of a difference in the time to response between the two arms (HR 0.81, 95% CI 0.46 to 1.41, p=0.45).\n\nParticipants eligible apart from criterion relating to falls. Of the 317 embedded trial participants, 33 (10.4%) were initially ‘almost’ eligible for the OTIS trial (handwritten: 17/159 [10.7%]; printed: 16/158 [10.1%]; difference 0.6%, 95% CI -6.2% to 7.3%). There was no evidence of a difference in the likelihood of participants being eligible on initial screen except for the risk factors for falling between the two groups (OR 1.06, 95% CI 0.52 to 2.19, p=0.87).\n\nParticipants eligible for trial. In total, 13 (4.1%) of the 317 embedded trial participants were eligible for the OTIS trial on their initial screening form (handwritten: 3/159 [1.9%]; printed: 10/158 [6.3%]; difference -4.4%, 95% CI -8.8% to -0.1%). There was weak evidence of a difference in the likelihood of participants being fully eligible on initial screen between the two groups (OR 0.28, 95% CI 0.08 to 1.05, p=0.06), in favour of the control group.\n\nParticipants who remained in the trial at 3 months post randomisation (return first three falls calendars). Of the 317 embedded trial participants, 10 (3.2%) remained in the OTIS trial 3 months post-randomisation (handwritten: 2/159 [1.3%]; printed: 8/158 [5.1%]; difference -3.8%, 95% CI -7.6% to 0%). There was some evidence of a difference in the proportion of embedded trial participants remaining in the main OTIS trial between the two groups in favour of the control group (unadjusted OR 0.24, 95% CI 0.05 to 1.14, p=0.07). When the logistic regression was adjusted for main trial allocation (which reduced the included sample size to 12) the size of the effect was similar but the confidence interval was much wider and the p-value larger (handwritten: 2/3 [66.7%]; printed: 8/9 [88.9%]; adjusted OR 0.23, 95% CI 0.01 to 5.93, p=0.37).\n\n\nDiscussion\n\nWe found that recipients of a personalised invitation letter, on which their name had been handwritten, were three times less likely to be randomised into the host OTIS trial, and this difference was statistically significant at the 10% level.\n\nFinding that personalisation did not increase recruitment to the OTIS trial provides a significant contribution to the limited literature on improving randomisation and recruitment to RCTs. The outcomes of the trial do not support the review by Edwards et al.8, which found that personalisation increased the odds of participants’ returning a questionnaire. However, not all of the studies included in the review observed an increase in response rate using personalisation. Moss and Worthern17 found that writing the potential participant’s name by hand significantly reduced response rates when compared to typing the name. The review by Edwards et al.8 included a wide range of studies within a wide range of contexts (e.g. teachers, students and individuals selected at random from the telephone directory). Moss and Worthern17 invited psychologists to provide their views on standardised assessments. They suggest that the reduction in response rates may have been due to handwriting being perceived as more personal but less professional. This supports Linsky’s theory that there are complex interactions between methods used to increase recruitment and the context in which they are used18. Our findings may also provide additional evidence for Linsky’s18 recommendation that in contexts requiring higher levels of confidentiality and professionalism, such as health research, personalisation may be contra-indicated.\n\nThere are some limitations to the study. Only a small number of potential participants were recruited and randomised to the OTIS study, indeed far fewer than anticipated, and so the embedded trial is severely underpowered. This reduces the reliability of our findings. This highlights the value of repeating this investigation with larger sample sizes and reviewing findings across similar studies. Potential participants were limited to individuals over the age of 65 years living in the community, as such the results are only applicable to this population. Further studies should substantiate the study results in other populations.\n\n\nConclusion\n\nOur findings that personalisation does not improve recruitment lend weight to the argument that methods to improve recruitment may well be context specific. Given the small sample size the results should be interpreted with caution and highlight the need to replicate and extend this work across larger studies and wider populations.\n\n\nData availability\n\nOpen Science Framework: OTIS Invitation Letter SWAT. https://doi.org/10.17605/OSF.IO/KGH4S16.\n\nThis project contains the underlying data in CSV and SAV format, with a variable key in CSV format.\n\nOpen Science Framework: OTIS Invitation Letter SWAT. https://doi.org/10.17605/OSF.IO/KGH4S16.\n\nThis project contains the following extended data:\n\nSupplementary File 1. Invitation letter for the Control Group.\n\nSupplementary File 2. Invitation letter for the Intervention Group.\n\n\nReporting guidelines\n\nOpen Science Framework: CONSORT checklist for study “Does handwriting the name of a potential trial participant on an invitation letter improve recruitment rates? A randomised controlled study within a trial”. https://doi.org/10.17605/OSF.IO/KGH4S16.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe OTIS study was funded by the National Institute for Health Research (NIHR) Health Technology Assessment (HTA) Programme (Programme grant number: 14/49/149). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. This SWAT was funded by York Trials Unit. The University of York is the study sponsor and has legal responsibility for the initiation and management of the trial (sponsor representative: Dr Michael Barber, Research and Enterprise Directorate, University of York, Ron Cooke Hub, Heslington, York, UK, YO10 5GE).\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\nThe authors would like to thank the embedded trial participants who returned trial recruitment documentation. This manuscript has been written by the authors on behalf of the OTIS Study Team. Sophie Boyes (York Teaching Hospital NHS Foundation Trust); Sarah Cockayne (University of York); Belen Corbacho (University of York); Shelley Crossland (Leicestershire Partnership NHS Trust); Avril Drummond (University of Nottingham); Caroline Fairhurst (University of York); Simon Gilbody (University of York); Catherine Hewitt (University of York); Sarah E Lamb (University of Oxford); Jennifer McCaffery (University of York); Alison Pighills (Mackay Base Hospital; Mackay Australia and James Cook University); Clare Relton (University of Sheffield); Sara Rodgers (University of York); and David J. Torgerson (University of York).\n\n\nReferences\n\nDjulbegovic B, Guyatt GH: Progress in evidence-based medicine: a quarter century on. Lancet. 2017; 390(10092): 415–23. PubMed Abstract \| Publisher Full Text\n\nBrownson RC, Fielding JE, Maylahn CM: Evidence-based public health: a fundamental concept for public health practice. Annu Rev Public Health. 2009; 30: 175–201. PubMed Abstract \| Publisher Full Text\n\nGreenhalgh T, Howick J, Maskrey N, et al.: Evidence based medicine: a movement in crisis? BMJ. 2014; 348: g3725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalters SJ, Bonacho Dos Anjos Henriques-Cadby I, Bortolami O, et al.: Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open. 2017; 7(3): e015276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaldwell PH, Hamilton S, Tan A, et al.: Strategies for increasing recruitment to randomised controlled trials: systematic review. PLoS Med. 2010; 7(11): e1000368. 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\nTreweek S, Pitkethly M, Cook J, et al.: Strategies to improve recruitment to randomised trials. Cochrane Database Syst Rev. 2018; 2: MR000013. PubMed Abstract \| Publisher Full Text\n\nEdwards PJ, Roberts I, Clarke MJ, et al.: Methods to increase response to postal and electronic questionnaires. Cochrane Database Syst Rev. 2009; (3): MR000008. PubMed Abstract \| Publisher Full Text\n\nScott P, Edwards P: Personally addressed hand-signed letters increase questionnaire response: a meta-analysis of randomised controlled trials. BMC Health Serv Res. 2006; 6: 111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCockayne S, Pighills A, Adamson J, et al.: Can occupational therapist-led home environmental assessment prevent falls in older people? A modified cohort randomised controlled trial protocol. BMJ Open. 2018; 8(9): e022488. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRelton C, Torgerson D, O'Cathain A, et al.: Rethinking pragmatic randomised controlled trials: introducing the \"cohort multiple randomised controlled trial\" design. BMJ. 2010; 340: c1066. PubMed Abstract \| Publisher Full Text\n\nRelton C, Bissell P, Smith C, et al.: South Yorkshire Cohort: a 'cohort trials facility' study of health and weight - protocol for the recruitment phase. BMJ Public Health. 2011; 11: 640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCockayne S, Adamson J, Clarke A, et al.: Cohort Randomised Controlled Trial of a Multifaceted Podiatry Intervention for the Prevention of Falls in Older People (The REFORM Trial). PLoS One. 2017; 12(1): e0168712. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLewis H, Adamson J, Atherton K, et al.: CollAborative care and active surveillance for Screen-Positive EldeRs with subthreshold depression (CASPER): a multicentred randomised controlled trial of clinical effectiveness and cost-effectiveness. Health Technol Assess. 2017; 21(8): 1–196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShepstone L, Lenaghan E, Cooper C, et al.: Screening in the community to reduce fractures in older women (SCOOP): a randomised controlled trial. Lancet. 2018; 391(10122): 741–7. PubMed Abstract \| Publisher Full Text\n\nMcCaffery J, Mitchell A, Fairhurst C, et al.: Does Handwriting the Name of a Potential Trial Participant on an Invitation Letter Improve Recruitment Rates? A randomised control trial within a trial. 2019.\n\nMoss VD, Worthen BR: Do personalization and postage make a difference on response rates to surveys of professional populations? Psychol Rep. 1991; 68(2): 692–4. Publisher Full Text\n\nLinsky AS: Stimulating responses to mailed questionnaires: A review. Public Opin Q. 1975; 39(1): 82–101. Publisher Full Text" }	[ { "id": "50004", "date": "18 Jun 2019", "name": "Martin C. Gulliford", "expertise": [ "Reviewer Expertise epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful trial within a trial to explore different invitation methods. The report clearly describes what was done and presents clear conclusions.\nComments:\nThe intervention appears to be under-theorised as a behaviour change intervention. It might be useful to cross reference the behavioural insights approach (e.g. publications by Anna Sallis) as well as using the TIDIER checklist.\n\nIn the analysis, the paper presents both a difference in proportions and an odds ratio, though the P value is not given for the former. The odds ratio appears to be considered the primary analysis, though the difference in proportions is more relevant. It is not clear that both analyses are needed.\n\nIn the Discussion, where it reads 'and this difference was statistically significant at the 10% level.' The ASA guidelines suggest that bright lines such as 0.1 or 0.05 should not be used. This P value does not appear to provide evidence of any real effect. https://www.amstat.org/asa/files/pdfs/P-ValueStatement.pdf\n\nMay want to reference the Cowie study.1\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "52960", "date": "29 Aug 2019", "name": "Miles D. Witham", "expertise": [ "Reviewer Expertise Clinical trials", "particularly for older people" ], "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 SWAT (Study within a Trial) is a useful addition to the literature on trials recruitment methodology, and is no less valuable for attempting to replicate a previously tested intervention.\nThe methods are appropriately described and there are links to the intervention and comparator letters as well as to the underlying data.\nThe numbers are small, but the results are reported appropriately and the small numbers are acknowledged in the discussion. Limitations are appropriately discussed and conclusions are appropriately cautious.\n\nPoints for consideration:\nI would like to know who actually wrote the participant name on the letters and produced the mailout packs; in particular, it needs to be clear if they had any role in the SWAT or the main trial beyond generating the mailout packs? This would reassure readers that the analysis was masked and that the allocation of packs did not influence any other study processes. A statement about who was masked (analysis team, main study team) and who wasn’t masked would also be helpful.\n\nThe choice of a 10% significance level is non-standard (but is not inappropriate given the small anticipated sample size) – please could the authors explain and justify this choice to readers though?\n\nRef 16 in the reference list is a reference to this paper, but without the hyperlink to the supplementary data to which the in-text reference seems to apply. Could this be fixed please?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-659
https://f1000research.com/articles/8-323/v1	22 Mar 19	{ "type": "Research Article", "title": "Young people in HIV care in Ukraine: a national survey on characteristics and service provision", "authors": [ "Galyna Kyselyova", "Violeta Martsynovska", "Alla Volokha", "Nataliya Nizova", "Ruslan Malyuta", "Ali Judd", "Claire Thorne", "Heather Bailey", "Galyna Kyselyova", "Violeta Martsynovska", "Alla Volokha", "Nataliya Nizova", "Ruslan Malyuta", "Ali Judd", "Claire Thorne" ], "abstract": "Background: Ukraine’s perinatally HIV-infected (PHIV) young people are ageing into adolescence/young adulthood and, alongside those with behaviourally-acquired infections (BHIV), require transitional and other support services. We aimed to map this population and policies/service provision at specialist HIV centres, to inform future service development. Methods: A national survey was conducted of 28 HIV/AIDS centres on number, characteristics (age group, HIV acquisition mode) and care setting (paediatric/adult) of 10-24 year olds in HIV care in each of 24 regions in January 2016. Information was collected on policies/service provision at each centre. Results: Of 13,286 young people aged 10-24 years registered for HIV care nationally in Ukraine in January 2016, 1,675 were aged 10-18 years. Three-quarters of ≤19 year olds were PHIV, while 72% of 20-24-year-olds had sexually-acquired infection. Five regions accounted for two-thirds of 10-18 year olds in paediatric and 85% of 19-24 year olds in adult services. In 2015, 97 young people transitioned from paediatric to adult services nationally, typically at 18 years although with flexibility in timing at 17/28 centres. At 27/28 centres, BHIV young people aged <18 years began their HIV care in paediatric services sometimes (5) or always (22). Transition support most commonly consisted of a joint appointment with paediatrician and adult doctor, and support from a psychologist/social worker (both at 24/28 centres). Only 5/28 centres offered routine HIV care during the evening or weekend, and availability of integrated sexual/reproductive health and harm reduction services was uneven. Of 16/28 centres selectively following-up patients who did not attend for care, 15 targeted patients in paediatric services. Conclusions: Heterogeneity in the population and in service availability at the main regional/municipal HIV/AIDS centres has implications for potential structural barriers to HIV care, and development of services for this group.", "keywords": [ "HIV", "youth", "adolescents", "Ukraine", "Eastern Europe", "transition", "perinatal HIV infection", "injecting drug use", "reproductive health", "harm reduction" ], "content": "Abbreviations\n\nART, antiretroviral therapy; BHIV, behaviourally HIV-infected; IDU, injecting drug use; PHIV, perinatally HIV-infected; PLHIV, people living with HIV; PWID, people who inject drugs\n\n\nIntroduction\n\nUkraine, a middle income country, has the second largest population of people living with HIV (PLHIV) in Europe, estimated at 238,0001. Important successes in prevention of mother-to-child transmission and paediatric treatment programmes are reflected, as in other settings, in the demographic characteristics of patients with perinatal HIV (PHIV), who are an ageing cohort. National data indicate that there were 3,014 HIV-infected children and young people aged <18 years in HIV care in Ukraine at the end of 20161. Around half of the PHIV population were estimated to be aged ≥10 years at this time with numbers transferring to adult care expected to peak in 2019–2026, based on an age at transfer of around 18 years2.\n\nIn terms of behaviourally-acquired HIV (BHIV) among young people in Ukraine, injecting drug use (IDU) has been a key driver of the epidemic3, including among marginalised youth4 as average age of IDU initiation is in the late teens5. In recent years, young people aged 15-24 years have accounted for a declining number and proportion of newly diagnosed HIV infections (12% in 2009 vs. 5.2% in 2016)1. However, an estimated 42% of PLHIV in Ukraine are undiagnosed6, and this proportion may be higher among young people, reflecting the specific barriers to services among young people who inject drugs (PWID) and their sexual partners7. Women accounted for 43% of new infections in 2015, but with a younger age of infection/diagnosis than men; a third of 15–49 year old women living with HIV were <30 years in 2015 vs. only 19% of men8.\n\nHIV services in Ukraine are delivered through specialist regional and municipal HIV/AIDS centres, in conjunction with local or satellite clinics to which antiretroviral therapy (ART) provision has been decentralised in recent years. Decentralised services are available mainly to adults, while children usually remain under the follow-up of a paediatrician at a specialist centre (personal communication, Galyna Kyselyova). HIV treatment and care are officially provided free-of-charge, however a system of unofficial payments may also apply, and support services are often provided by non-governmental organisations. Around two-thirds of diagnosed individuals linked to care are on ART.\n\nAdolescence and young adulthood are periods of vulnerability regarding access to and retention in healthcare services for chronic conditions, including HIV, due to a range of factors including increased risk-taking behaviours, challenges around transferring to and navigating adult-oriented health systems, managing stigma and disclosure, and emergence of mental health problems9,10. Findings from high income settings have indicated that PHIV young people are at elevated risk of poor ART adherence, virological failure and deterioration of health and loss to follow-up during adolescence and transition to adult care11–14, although UK data show substantial improvements over calendar time and improved CD4 trajectory in some groups post-transition15,16. The few studies to date evaluating different service delivery models for HIV-positive young people indicate the importance of clinic accessibility, integrated care and peer support, as well as the potential impact of individual-level interventions such as financial incentives for clinic attendance and treatment adherence17,18. However, quality of evidence regarding models of care for young people is poor, and the success of different models is likely to be highly setting-specific, while most studies are from high income settings.\n\nReforms of Ukraine’s healthcare system are ongoing and include development of more patient-centred, outcome-oriented models of care19. However, currently there is little evidence to guide the development of policies, care and support programmes specific to young PLHIV, despite evolving needs for services. The objectives of this study were to describe the contemporary population of young people aged 10–24 years receiving HIV care in Ukraine by mode of HIV acquisition, type of care (paediatric vs. adult) and region, and to document current service provision and local policies at specialist HIV centres in order to inform future service development.\n\n\nMethods\n\nIn April and May 2016, a paper-based questionnaire was sent to 24 regional HIV/AIDS centres and four large municipal HIV/AIDS centres (Kryvy Rih and Dnipro city centres in Dnepropetrovsk region and Bila Tserkva and Kiev city centres in Kiev region) in collaboration with the Public Health Center of the Ministry of Health of Ukraine. These centres were surveyed because they collate data on all people registered for HIV care nationally, within each of the 24 regions (oblasts), including those followed-up at local or satellite clinics.\n\nThe questionnaire requested the number of 10–24 year olds (young people, according to WHO’s definition20) receiving HIV care within the region on 1 January 2016. Based on an age of transfer to adult care of 18 years, the questionnaire requested the number of young people in paediatric and adult services in two age groups: 10–18 years and 19–24 years. To explore service needs in more depth, the number of young people by mode of HIV acquisition was disaggregated according to three age groups: 10–14 years, 15–19 years and 20–24 years, to accommodate WHO definition of adolescence (10–19 years)20 and facilitate comparison with national figures. Additional questions were on policies around transition, provision of psychological support, sexual and reproductive health and harm reduction services and practices around loss to follow-up at each regional/municipal centre. Responses were provided by a paediatrician at each centre or another member of staff with knowledge of models of care provided to young people.\n\nTo obtain an estimate of the total number of young people aged 10–24 years in HIV care nationally, we summed the regional totals. This national estimate was complete with the exception of the temporarily uncontrolled territories of the Donetsk and Lugansk regions (partial data available) and the Russian-occupied territories of The Crimea. To identify possible double-counting of young people registered simultaneously in paediatric and adult services within the same region, we compared the total across both services with the regional total by mode of HIV acquisition. Where the second total was smaller, we summed the 10–18 year olds registered in adult services and the 19–24 year olds registered in paediatric services, to reach an estimate of the maximum number double-counted across paediatric and adult services.\n\nData were entered into a REDCap database. Descriptive analyses were conducted in STATA version 13 (Stata Corp LP, College Station USA).\n\nAs a service evaluation, this survey did not require ethics approval, but was approved by the Ministry of Health. Individual patient data were not collected and consent was therefore not required.\n\n\nResults\n\nAll 28 centres responded; questionnaires were completed by paediatric/adult infectious diseases doctors (n=23), epidemiologists (n=2), an HIV/AIDS centre head (n=1), immunologist (n=1), and nurse (n=1).\n\nNationally, a total of 13,286 young people aged 10–24 years were registered for HIV care (paediatric and adult services) in January 2016, of whom 43 may have been registered simultaneously in both paediatric and adult services in the same region. Of the total 13,286, 13% (1,675) were aged 10–18 years, a group making up 94% (1418/1505) of those in paediatric services and 2% (257/11781) of those in adult services. The median number of 10–18 year olds registered in paediatric services in each region was 31 (range 1, 212) while that for 19–24 year olds in adult care was 101 (range 0, 3632). Dnepropetrovsk, Kiev, Mykolaiv and Odessa regions had amongst the largest numbers of patients in both age groups; along with Chernivtski, these regions accounted for almost two-thirds (65%, 917/1418) of 10–18 year olds in paediatric care (Figure 1a) and with Donetsk they accounted for 85% (9849/11,524) of 19–24 year olds in adult HIV services (Figure 1b). Overall, 839 10–24 year olds were newly registered for HIV care in 2015, 759 of whom were aged 19–24 years.\n\nMaps showing (a) number of 10–18 year olds in paediatric HIV care and (b) number of 19–24 year olds in adult HIV care by region, on 1 January 2016. Area of circle is proportional to number of young people. Note different scales for maps (a) and (b). Name of region was included for centres with (a) n≥100 10–18 year olds in paediatric services and (b) n≥800 19–24 year olds in adult services. Maps were adapted from original versions created by Aleksandr Grigoryev, available at https://commons.wikimedia.org/w/index.php?curid=28880433 under a CC0 license.\n\nTable 1 shows mode of HIV acquisition of young people in HIV care by age group. Overall 13% (1663/13060) were adolescents (10–19 years), most of whom were perinatally infected. There were 210 PHIV young people aged 20–24 years in HIV care at the beginning of 2016, accounting for 1.8% of this age group and 14% of the PHIV population overall. Among 15–19 year olds, 43% (294/691) had sexually-acquired HIV infection, increasing to 72% (8183/11397) in those aged 20–24 years. Young PWID accounted for a quarter of the 20–24 year old age group.\n\n†This total is 226 fewer than the total 13286 given in text due to unknown /unreported mode of HIV acquisition for some young people, and potential double-counting of up to 43 patients registered with both paediatric and adult services simultaneously.\n\nIn total 97 patients transitioned from paediatric to adult care across 13 regions during 2015; 66 in two regions (Dnepropetrovsk and Odessa). In 11/28 centres, transition always took place at 18 years, in two centres it could sometimes be postponed depending on the paediatric caseload, while in 15 centres transition could always be postponed if necessary; of the latter group of 15 centres, there was no formal age limit for transfer in eight, while the maximum age limit ranged up to 24 years in the remainder. At the 17 centres with flexible policies, possible reasons for postponement of transfer included lack of maturity or independence of young person (n=15), young person’s request (n=11), poor support at home (n=6), poor adherence (n=3), cognitive problems (n=3), poor health (n=1), and financial concerns related to payment for tests or drugs in adult services (n=1).\n\nMost (22/28) centres reported that BHIV young people aged <18 years began their HIV care in paediatric services while in five centres they sometimes initiated care in adult services depending on factors such as adequate maturity, pregnancy, wish to attend adult services, or availability of a paediatrician; one centre reported always registering BHIV young people in adult HIV services.\n\nTable 2 shows the number of centres offering each of four aspects of transition support. Most (24/28) centres offered young people a joint appointment with paediatrician and adult doctor as part of the transition process, with specific support from a psychologist or social worker also available at most centres surveyed. Only three centres reported that young people continued to see a paediatrician for a period after transfer to adult care.\n\n† One centre was missing data on transfers in 2015 and is included in the “overall” column only\n\nOf the 28 centres, five centres offered a weekend or evening clinic (for four this was at least weekly) while two centres reported out-of-hours services for emergencies only (e.g. post-exposure prophylaxis). All centres provided a “walk-in” service without an appointment during opening hours, either for standard follow-up (n=27) or only for urgent care (n=1).\n\nFigure 2 shows the proportion of centres surveyed offering each of six types of service/support in HIV care. Condoms were freely available at half of the centres and other contraceptives at four centres. Testing and treatment for sexually transmitted infections and psychological support were quite widely available; however, the three centres without psychologist support were in regions with some of the highest numbers of young people in HIV care (collectively 7287 of the 13286 10–24 year olds in HIV care nationally). Out of 26 centres, 17 reported offering a support group, and at six of these 17 centres a group was provided specifically for young people. Eleven centres had harm reduction services available to PWID routinely (n=8) or by referral (n=3). Regarding contributions towards the cost of HIV care in adult services, all 28 centres indicated that ART and general HIV care were free-of-charge, but seven centres indicated patient contributions to costs associated with blood tests (n=5), vaccinations (n=4) and/or general equipment /supplies (n=4). Only five centres offered financial support for travel costs to attend appointments.\n\nOverall, 25/28 centres provided appointment reminders for patients, mainly by phone call (23/25); only one centre reported using SMS and none used email. Twelve centres reported making contact with all patients who missed one follow-up, while 16 centres indicated a selective policy, mostly targeting those on ART (n=14), children or young people in paediatric services (n=15), and/or patients of heightened clinical concern – e.g. those who were pregnant, with low CD4 count, a recently changed ART regimen or preparing to start ART. Patients missing follow-up were most commonly contacted by a doctor (21/28) or nurse (24/28) at the HIV/AIDS centre, a social worker (20/28), a local doctor (13/28) or in one case a psychologist.\n\n\nDiscussion\n\nResults from this national survey show that of around 13,300 10–24 year olds in HIV care in Ukraine at the beginning of 2016, around 86% (11,400) were aged 20–24 years, with 72% of this age group having sexually-acquired HIV infection. Smaller sub-groups include over 3000 young PWID and increasing numbers of PHIV youth entering adult care. Our survey results indicated that 759 19–24 year olds were newly registered for HIV care in 2015. It is not possible to determine what proportion of all new diagnoses these young people represented (and therefore the proportion linked to care), because national figures are not disaggregated by age group. However, 26% of diagnosed individuals overall are currently unlinked to HIV care1.\n\nAs expected, young people aged ≤18 years were predominantly cared for in paediatric services. The number of young people transitioning from paediatric to adult care was fairly small – 97 in 2015 – but set to increase year on year. Although transition typically occurs at around 18 years, a range of policies were in place nationally, with some flexibility regarding timing of transition at 17 of 28 centres. This allowed clinicians to be responsive to young people’s developmental maturity and need for continuity of care, which may be important to their feelings of preparedness for transfer and trust in their healthcare provider21. Joint appointments with the paediatrician and adult doctor were part of transition processes alongside psychosocial support in most centres. The close involvement of paediatric and adult staff during transition is recommended in USA guidelines10 and may help young people to overcome barriers to transition which relate to fears of changing relationships with healthcare providers and new systems in adult services22. However, the evidence base for different transition models in improving outcomes is currently lacking. The recent decentralisation of HIV services for adults in Ukraine means that transfer to adult care may be increasingly accompanied by a change in location of care as well as provider; the potential impact of this on retention needs to be examined, along with implications for models of transition support.\n\nMost BHIV young people <18 years nationwide start their HIV care in paediatric services. Paediatricians may be better equipped to support adolescents in HIV care and treatment programmes through a greater understanding of adolescent development and behaviour23. However, given the usual age of transfer of 18 years, these young people are likely to lack continuity in their healthcare provider during their first years of HIV care and will navigate transition to adult care with less established relationships with paediatric healthcare providers than their PHIV counterparts. BHIV young people may experience complex barriers to care related, for example, stigma and concerns around confidentiality, which are reflected in longer delays linking to HIV services than is average for older BHIV adults24.\n\nThe WHO recommends co-location of HIV care with other health services relevant for key populations (e.g. sexual and reproductive health, drug dependence services)25 to improve accessibility of services, which is a key component of youth friendly healthcare. We found uneven availability of services integrated with HIV care at the regional and municipal HIV/AIDS centres included in our survey. Only around half of centres offered free condoms. Other sexually transmitted infections are prevalent26 and condoms are also a main method of family planning in this population27, reflected in the low availability of other forms of contraception as part of HIV care. The 14 centres with free condoms available were in regions with larger caseloads (collectively accounting for 75% of 10–24 year olds in HIV care). Similarly, harm reduction services for PWID were available at 11 centres, located in regions with 86% of young PWID in HIV care nationally, indicating some targeting of services. However, services may not be available to all of the young PLHIV in these regions; for example, harm reduction services were available at only one of the three centres surveyed in each of Kiev and Dnepropetrovsk regions.\n\nFinancial support towards transport costs was not available at most HIV centres surveyed. Transport costs are a key potential structural barrier to healthcare, sometimes addressed by clinicians on an informal basis in Ukraine (personal communication, Galyna Kyselyova), and may be compounded by informal costs for some aspects of healthcare (e.g. blood tests). Decentralisation of ART provision may minimise transport-related barriers for young people entering adult HIV care. Routine HIV care was available at almost all centres without an appointment, however only five centres offered services during the evenings or weekends. Availability of flexible care at times which minimise the need to miss school, college or work is an important aspect of age-appropriate care as defined by youth21 and evening clinics have been associated with better retention in care among HIV-positive young people across 12 sites in the USA28. Over half of centres reported a selective policy with regards contact with patients who missed an appointment, with 15/16 of these centres indicating that they would target those in paediatric services. The time of transition to adult care may therefore coincide with diminishing support and follow-up, which is of concern given evidence from other settings of challenges retaining adolescents and young adults in care12,29. Use of mobile technologies was almost completely absent, but may be a useful means to improve ART adherence and support in HIV care for adolescents30.\n\nSurvey results indicated that two-thirds of 10–18 year olds in paediatric HIV care were in just five of the 24 regions, with a similar pattern in 19–24 year olds, reflecting the concentration of the epidemic in the South and East of the country, including some regions also affected by conflict and disruption of HIV and related services in recent years31. While services targeted to these regions will achieve the greatest coverage, different support may be needed in those regions with lower caseloads, tailored to the more limited experience in the clinical and psycho-social care of PHIV children and adolescents (for example, in disclosing to PHIV young people their HIV status) and fewer peer support opportunities.\n\nPeer support opportunities for PHIV young people will increase in coming years as this group reach adolescence and young adulthood in greater numbers, and youth-oriented initiatives (such as those led by community-based organisation Teenergizer) are needed to shape support for the needs of this group alongside BHIV youth. PHIV and BHIV young people’s experiences of and barriers to HIV care need to be explored in the context of Ukraine’s healthcare reforms towards more patient-centred services, and we recently undertook a survey of PHIV and BHIV young people at two HIV/AIDS centres which investigated these topics, with work ongoing in this area. Importantly, the first cohort of PHIV young people reaching adulthood have different clinical and social characteristics to younger groups – for example, are more likely to be in extended family (vs parental) care – with implications for the level and type of support they may need as they reach adulthood.\n\nThere are a number of limitations to this work. We estimated that up to 43 young people may have been double-counted due to simultaneous registration with paediatric and adult services in one region, but could not estimate the number counted by ≥1 region (e.g. due to internal migration or living with extended family members). Figures from the Russian-occupied territories are incomplete. We did not disaggregate young people with sexually-acquired HIV infection into men who have sex with men (MSM) and those with heterosexually-acquired infection, due to the particular vulnerability of MSM in Ukraine which is linked with their under-reporting and misclassification in official figures32. Although the number of young people in HIV care in each region gives a complete national picture, we could not accurately estimate their coverage with the services provided at the 28 regional and municipal HIV/AIDS centres included in this survey, because ART and HIV care have been decentralised to smaller local clinics in recent years; services are likely to be less readily available at smaller clinics, particularly for key populations.\n\n\nConclusions\n\nThere is substantial heterogeneity in young PLHIV in Ukraine by geography, age and mode of HIV acquisition, and in services at regional HIV/AIDS centres. The number of PHIV young people requiring care during adolescence and young adulthood will continue to increase. Uneven availability of integrated services, financial barriers and lack of evening or weekend services may pose structural barriers to HIV care for both PHIV and BHIV young people and further work is needed to understand these. The results of this study underscore the importance of including services for adolescents in Ukraine’s comprehensive unified Clinical Protocol on HIV/AIDS, with care models focussed on sustainability, effectiveness and scale up of decentralization of services in the context of wider healthcare reforms in Ukraine.\n\n\nData availability\n\nOpen Science Framework: Young people in HIV care in Ukraine: a national survey on characteristics and service provision. https://doi.org/10.17605/OSF.IO/XS7ZE33.\n\nThis project contains the following underlying data:\n\n- Ukraine HIV centre survey data.csv\n\n- Ukraine HIV centre survey data dictionary.csv\n\nOpen Science Framework: Young people in HIV care in Ukraine: a national survey on characteristics and service provision. https://doi.org/10.17605/OSF.IO/XS7ZE33.\n\nThis project contains the following extended data:\n\n- Final policy survey_English.docx\n\n- Final policy survey_Russian.docx", "appendix": "Grant information\n\nFunding for this project was made possible by a CIPHER grant from the International AIDS Society. The views expressed in written materials or publications do not necessarily reflect the official policies of the International AIDS Society. This research was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.\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\nWith grateful thanks to all survey respondents.\n\nStudy data were managed using REDCap electronic data capture tools hosted at University College London34. REDCap (Research Electronic Data Capture) is a secure, web-based application designed to support data capture for research studies, providing: 1) an intuitive interface for validated data entry; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for importing data from external sources.\n\n\nReferences\n\nState Institution Public Health Center of the Ministry of Health of Ukraine: HIV Infection in Ukraine Information Bulletin, No. 47. Accessed June 2017. 2017. Reference Source\n\nBailey H, Kiseleva G, Malyuta R, et al.: Forecasting future burden and need for transitional services for young people with perinatally-acquired HIV in Ukraine, TUPEC167. In: International AIDS Conference: 2016; Durban; 2016.\n\nJolley E, Rhodes T, Platt L, et al.: HIV among people who inject drugs in Central and Eastern Europe and Central Asia: a systematic review with implications for policy. BMJ Open. 2012; 2(5): pii: e001465. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNerlander LM, Zapata LB, Yorick R, et al.: Behaviors Associated With a Risk of HIV Transmission From HIV-Positive Street Youth to Non-Street Youth in Ukraine. Sex Transm Dis. 2015; 42(9): 513–520. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMakarenko I, Ompad DC, Sazonova Y, et al.: Trends in Injection Risk Behaviors among People Who Inject Drugs and the Impact of Harm Reduction Programs in Ukraine, 2007-2013. J Urban Health. 2017; 94(1): 104–114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNAIDS: Prevention Gap Report. Accessed August 2016. 2016. Reference Source\n\nDelany-Moretlwe S, Cowan FM, Busza J, et al.: Providing comprehensive health services for young key populations: needs, barriers and gaps. J Int AIDS Soc. 2015; 18(2 Suppl 1): 19833. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nState Institution \"Ukrainian Center for Socially Dangerous Disease Control of the Ministry of Health of Ukraine\": HIV Infection in Ukraine Information Bulletin, No. 45. Accessed March 2017. 2016. Reference Source\n\nBernays S, Jarrett P, Kranzer K, et al.: Children growing up with HIV infection: the responsibility of success. Lancet. 2014; 383(9925): 1355–1357. PubMed Abstract \| Publisher Full Text\n\nCommittee On Pediatric Aids: Transitioning HIV-infected youth into adult health care. Pediatrics. 2013; 132(1): 192–197. PubMed Abstract \| Publisher Full Text\n\nAgwu AL, Fairlie L: Antiretroviral treatment, management challenges and outcomes in perinatally HIV-infected adolescents. J Int AIDS Soc. 2013; 16: 18579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyscavage P, Macharia T, Patel D, et al.: Linkage to and retention in care following healthcare transition from pediatric to adult HIV care. AIDS Care. 2016; 28(5): 561–565. PubMed Abstract \| Publisher Full Text\n\nKakkar F, Van der Linden D, Valois S, et al.: Health outcomes and the transition experience of HIV-infected adolescents after transfer to adult care in Québec, Canada. BMC Pediatr. 2016; 16: 109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeilan AM, Karalius B, Patel K, et al.: Association of Risk of Viremia, Immunosuppression, Serious Clinical Events, and Mortality With Increasing Age in Perinatally Human Immunodeficiency Virus-Infected Youth. JAMA Pediatr. 2017; 171(5): 450–460. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollins IJ, Foster C, Tostevin A, et al.: Clinical Status of Adolescents with Perinatal HIV at Transfer to Adult Care in the UK/Ireland. Clin Infect Dis. 2017; 64(8): 1105–1112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJudd A, Collins IJ, Parrott F, et al.: Growing up with perinatal HIV: changes in clinical outcomes before and after transfer to adult care in the UK. J Int AIDS Soc. 2017; 20(Suppl 3): 21577. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJudd A, Sohn AH, Collins IJ: Interventions to improve treatment, retention and survival outcomes for adolescents with perinatal HIV-1 transitioning to adult care: moving on up. Curr Opin HIV AIDS. 2016; 11(5): 477–486. PubMed Abstract \| Publisher Full Text\n\nMacPherson P, Munthali C, Ferguson J, et al.: Service delivery interventions to improve adolescents' linkage, retention and adherence to antiretroviral therapy and HIV care. Trop Med Int Health. 2015; 20(8): 1015–1032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealth Strategic Advisory Group: National Health Reform Strategy for Ukraine, 2015-2020. Accessed February 2017. 2015. Reference Source\n\nWHO: recognising adolescence. Accessed November 2017. Reference Source\n\nAmbresin AE, Bennett K, Patton GC, et al.: Assessment of youth-friendly health care: a systematic review of indicators drawn from young people's perspectives. J Adolesc Health. 2013; 52(6): 670–681. PubMed Abstract \| Publisher Full Text\n\nSharma N, Willen E, Garcia A, et al.: Attitudes toward transitioning in youth with perinatally acquired HIV and their family caregivers. J Assoc Nurses AIDS Care. 2014; 25(2): 168–175. PubMed Abstract \| Publisher Full Text\n\nLee L, Rand CS, Ellen JM, et al.: Factors informing HIV providers' decisions to start antiretroviral therapy for young people living with behaviorally acquired HIV. J Adolesc Health. 2014; 55(3): 358–365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiriazova TK, Postnov OV, Perehinets IB, et al.: Association of injecting drug use and late enrolment in HIV medical care in Odessa Region, Ukraine. HIV Med. 2013; 14 Suppl 3: 38–41. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organisation: Consolidated guidelines on HIV prevention, diagnosis, treatment and care for key populations. 2014, revised 2016. Reference Source\n\nAebi-Popp K, Bailey H, Malyuta R, et al.: High prevalence of herpes simplex virus (HSV)- type 2 co-infection among HIV-positive women in Ukraine, but no increased HIV mother-to-child transmission risk. BMC Pregnancy Childbirth. 2016; 16(1): 94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBailey H, Thorne C, Semenenko I, et al.: Cervical screening within HIV care: findings from an HIV-positive cohort in Ukraine. PLoS One. 2012; 7(4): e34706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee L, Yehia BR, Gaur AH, et al.: The Impact of Youth-Friendly Structures of Care on Retention Among HIV-Infected Youth. AIDS Patient Care STDS. 2016; 30(4): 170–177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgwu AL, Lee L, Fleishman JA, et al.: Aging and loss to follow-up among youth living with human immunodeficiency virus in the HIV Research Network. J Adolesc Health. 2015; 56(3): 345–351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organisation: HIV and adolescents: guidance for HIV testing and counselling and care for adolescents living with HIV. Recommendations for a public health approach and considerations for policy-makers and managers. Geneva, Switzerland; 2013. Reference Source\n\nKazatchkine M: Towards a new health diplomacy in eastern Ukraine. Lancet HIV. 2017; 4(3): e99–e101. PubMed Abstract \| Publisher Full Text\n\nČakalo JI, Božičević I, Vitek C, et al.: Misclassification of men with reported HIV infection in Ukraine. AIDS Behav. 2015; 19(10): 1938–1940. PubMed Abstract \| Publisher Full Text\n\nBailey H: Young People in HIV Care in Ukraine: A National Survey on Characteristics and Service Provision, DOI 10.17605/OSF.IO/XS7ZE. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/XS7ZE\n\nHarris PA, Taylor R, Thielke R, et al.: Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform. 2009; 42(2): 377–381. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "46108", "date": "16 Apr 2019", "name": "Lee Fairlie", "expertise": [ "Reviewer Expertise Paediatric", "Adolescent and Maternal HIV", "TB and vaccine preventable diseases. This manuscript provides important details specifically relevant to those practising in that Country and similar countries regarding programming and improvements that could be made to services." ], "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 manuscript. This is a well written manuscript describing the population of adolescents and young people living with HIV in the Ukraine, based on a National survey. This manuscript highlights specific characteristics of this population and it is very interesting to see how this data may differ from other parts of the world for example sub Saharan Africa, where the characteristics of PLHIV in this age group is quite different. A few suggestions which could strengthen the manuscript are included below. These are for minor consideration by the authors:\nConsider using vertical transmission and horizontal transmission instead of \"behaviourally-acquired\" as this term carries substantial stigma.\n\nIntroduction:\n3rd paragraph, last line, it is reported that 2/3 of PLHIV are on treatment. It is worth elaborating on this and possible reasons in the test and treat era.\n\nResults:\nIn the 2nd paragraph, where age ranges and medians are given, suggest adding IQR, as the range is so vast that this data is difficult to interpret. Suggest adding whether any clinical criteria were considered when transition was decided for example VL suppression, clinical stability (no opportunistic infections, malignancies), pregnancy etc. Under \"Support services and follow-up....\" separate and discuss separately STIs and psychosocial support, as these are obviously quite different needs and both important. It is really interesting to see data on the mode of HIV acquisition—is this information routinely recorded in the notes? A limitation may be that this data may not be accurate and there may be situations where it is difficult to ascertain, for example, if maternal status is unknown or +, a young person who presents late for the first time may have acquired HIV vertically or horizontally.\n\nDiscussion:\nSuggest adding the above to the discussion. In the financial support paragraph, suggest also discussing differentiated models of care and also Youth Care Clubs or similar models where less facility-based contact is required. Also consider adding some text regarding the differences between this cohort and others, for example, in SSA .\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": "4615", "date": "14 May 2019", "name": "Heather Bailey", "role": "Author Response", "response": "Thank you for the helpful comments. We have removed “behaviourally-acquired HIV” as a term and replaced with horizontally-acquired. We have edited the line about 2/3 of PLHIV being on treatment to specify that this was in 2016 (it was similar in 2017, more recent data not yet available). Results – we have added IQR as suggested. Clinical criteria for postponing transition were captured with the option “young person is in poor health” which was selected by one centre. The section about STIs and psychosocial support has been edited to separate these out. Data on mode of HIV acquisition is routinely reported, however there is evidence of misclassification of MSM as having heterosexually-acquired HIV infection which we describe in the limitations section – we have also added some text to say that “mode of HIV acquisition may be misclassified in other ways (for example due to late diagnosis of a PHIV young person).” We agree that decentralisation of care is an important point for overcoming financial barriers to travelling to the regional/municipal HIV/AIDS centre and mention this." } ] }, { "id": "47442", "date": "02 May 2019", "name": "Qiang Xia", "expertise": [ "Reviewer Expertise Epidemiology", "Public Health", "Surveillance", "Survey methods", "HIV", "STD", "TB" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe the findings from a national survey of 28 HIV/AIDS centres in Ukraine on the characteristics of young people (10-24 year olds) living with HIV (PLHIV), the policies around the transition from paediatric to adult care, and services provided at these HIV/AIDS centres for young PLHIV. Overall, the methodology is sound, and the data are clearly presented.\n\nAbstract The writing could be tightened, especially in the results section, where only major findings should be presented.\n\nKeywords Please limit the number of keywords. The authors may consider removing, “injecting drug use,” “reproductive health,” and “harm reduction.”\n\nAbbreviations Please make the abbreviations consistent with those in the text, “PHIV, perinatally HIV-infected” vs. “patients with perinatal HIV (PHIV)” in the text, and “BHIV, behaviourally HIV-infected” vs. “behaviourally-acquired HIV (BHIV)” in the text.\n\nMethods Please make it clear that there is a total of 28 HIV/AIDS centres, and all of them were included in the survey.\n\nResults “Out of 26 centres, 17 reported offering a support group.” Why is it 26 centres not 28 centres? If two centres did not respond to this specific question, state it clearly in the sentence.\n\nTable 2 It is not informative to compare types of transition support between centres with no transfers in 2015 and centres with transfers in 2015. If the authors like, they may make comparisons between 24 regional centres and four municipal centres, or by region (e.g., East and West), the size of the patient population, or other characteristics.\n\nFigure 2 The bars are not at the same height, because of missing data. The authors may consider adding a category, and showing the data in four categories: 1) service available at all appointments, 2) service available by special arrangement or referral, 3) service not available, and 4) data not available.\n\nDiscussion “However, 26% of diagnosed individuals overall are currently unlinked to HIV care.” The meaning of the word, “unlinked,” is not clear. If the authors are talking about linkage to care, the sentence should be, “26% of diagnosed individuals overall have never been linked to HIV care.” If the authors are talking about retention in care, the sentence should be, “26% of diagnosed individuals overall are currently not retained in HIV care.”\n\nThe analysis unit of the study is HIV/AIDS centre, e.g., the number and proportion of HIV/AIDS centres with STI testing and treatment services. The analysis is valid, but readers and policy makers are also interested, if not more interested, in the analysis in which patient is the analysis unit, e.g., the number and proportion of patients with access to STI testing and treatment services at HIV/AIDS centres. The authors may consider adding the analysis with patient as the analysis unit in the Results, or listing lack of such an analysis as a limitation in 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? Yes", "responses": [ { "c_id": "4616", "date": "14 May 2019", "name": "Heather Bailey", "role": "Author Response", "response": "Thank you for the helpful comments. We have made some clarifications in the methods and results as suggested. In table 2, we made the decision to compare support services at centres with recent transfers and those without to identify any differences in reports of support that may exist in policy vs practice. Figure 2 – we have added a footnote to explain that bars differ in height due to missing data. Discussion – thank you for the point about the term ‘unlinked’ – in this context we meant that 26% have never been linked to care and have made this edit. We are not able to comment on the coverage of individuals with STI testing and treatment (and other) services because of the decentralisation of ART and HIV services to smaller local clinics in recent years. The survey could tell us the total number of young people in each region and availability of each type of support at the regional/municipal centre, but not the precise coverage of young people with these services as part of their HIV care (last paragraph of the discussion)." } ] } ]	1	https://f1000research.com/articles/8-323
https://f1000research.com/articles/7-1757/v1	06 Nov 18	{ "type": "Research Article", "title": "Genomic architecture differences at the HTT locus underlie symptomatic and pre-symptomatic cases of Huntington’s disease.", "authors": [ "Matthew Salter", "Ryan Powell", "Jennifer Back", "Francis Grand", "Christina Koutsothanasi", "Jayne Green", "Ewan Hunter", "Aroul Ramadass", "Jurjen Westra", "Alexandre Akoulitchev", "Matthew Salter", "Ryan Powell", "Jennifer Back", "Francis Grand", "Christina Koutsothanasi", "Jayne Green", "Ewan Hunter", "Aroul Ramadass", "Jurjen Westra" ], "abstract": "Background: Huntington’s disease (HD) is a progressive neurodegenerative condition that causes degeneration of neurons in the brain, ultimately leading to death. The root cause of HD is an expanded trinucleotide cytosine-adenine-guanine (CAG) repeat in the “huntingtin gene” (HTT). While there is a rough correlation between the number of CAG repeats and disease onset, the development of clinical symptoms can vary by decades within individuals and little is known about this pre-symptomatic phase. Methods: Using peripheral blood samples from HD patients and healthy controls we used EpiSwitch™, a validated high-resolution industrial platform for the detection of chromosome conformations, to assess chromatin architecture in the immediate vicinity of the HTT gene. We evaluated chromatin conformations at 20 sites across 225 kb of the HTT locus in healthy controls, verified symptomatic HD patients (CAG, n>39) and patients with CAG expansions who had not yet manifested clinical symptoms of HD. Results: Discrete chromosome conformations were observed across the patient groups. We found two constitutive interactions (occurring in all patient groups) and seven conditional interactions which were present in HD, but not in healthy controls. Most important, we observed three conditional interactions that were present only in HD patients manifesting clinical symptoms (symptomatic cases), but not in presymptomatic cases. Of the patients in the symptomatic HD cohort, 86% (6 out of 7) demonstrated at least one of the specific chromosome conformations associated with symptomatic HD. Conclusion: Our results provide the first evidence that chromatin architecture at the HTT locus is systemically altered in patients with HD, with conditional differences between clinical stages. Given the high clinical need in having a molecular tool to assess disease progression in HD, these results strongly suggest that the non-invasive assessment of chromosome conformation signatures can be a valuable addition to prognostic assessment of HD patients.", "keywords": [ "Huntington’s disease", "epigenetics", "chromosome conformation signature", "chromatin architecture" ], "content": "Introduction\n\nHuntington’s disease (HD) is a neurodegenerative condition characterized cellularly by the loss of neurons in the basal ganglia and clinically by uncontrolled movements, emotional problems, and loss of cognition1. HD is an autosomal dominantly inherited disorder and although prevalence rates range widely depending on geography and ethnicity, it is thought to affect more than 50,000 people in the United States and Europe alone2. The underlying genetic cause is a trinucleotide CAG expansion in the huntingtin gene (HTT), discovered as a genetic marker by James Gusella from Massachusetts General Hospital in 1983, which results in the production of a mutant huntingtin protein (mHTT) with a toxic poly-glutamine (polyQ) tract3,4. However, despite decades of research and clinical trials, no successful therapy has yet been developed. The “typical” onset of HD is between the ages of 40–50 years, but up to 15% of cases have very late onset and don’t show clinical symptoms until after the age of 60 years5. In a recent meta-analysis of studies investigating cases of late-onset HD (LoHD, defined as onset after 60 years of age), more than 90% of patients had CAG repeat lengths of ≤446. One of the more interesting observations in HD is that while there is a well-known correlation between the length of the polyQ repeat tract and the onset and severity of the disease, there is substantial variability within individual patients. For example, in patients with mid-range repeat lengths (defined here as between 40 and 50), disease onset can vary by 60 years in any individual patient5. This means that many patients who are carriers of polyQ tracts that predispose to the development of the disease can live for decades in a “presymptomatic” state7. What controls the onset of clinical symptoms remains currently unknown, and complicates the prognostic evaluation of HD patients.\n\nAlthough historically considered a monogenic disease, extensive research into the underlying pathology of HD suggests that the mechanisms leading to disease onset and progression are more complex than originally thought. Many different technologies have been used to look at the molecular changes underlying disease progression in HD, including gene expression, proteomics, metabolomics, network analysis, genomics and single nucleotide polymorphism (SNP) profiling8–13. As HD is considered a paradigm of a disease characterized by epigenetic dysregulation, more recently epigenetic approaches have emerged as a promising new tool for assessing pathology-related changes14. Most epigenetic studies in HD have focused on looking at genome-wide histone modifications (acetylation, methylation) or histone modifications at specific loci related to HD15–18. While these approaches have provided interesting insight into the disease, they have yielded often conflicting results and shown inconsistencies between mouse models and human disease14,19,20. Indeed, due to the global nature of these types of epigenetic analyses, they may lack the sensitivity to discriminate the subtler changes associated with disease onset and progression. As such, a consensus picture of epigenetic deregulation in HD using histone modification readouts has yet to materialize. However, not all molecular mechanisms associated with epigenetic regulation have been assessed in the context of HD. An important aspect of epigenetic regulation is at the level of 3-dimensional (3D) genomic architecture21.\n\nThe 3D organization of the genome reflects the heterogeneous effects of external environmental cues and inputs, and can be empirically measured by the assessment of chromosome conformations or when several conformations are measured concomitantly, a chromosome conformation signature (CCS)21. CCSs can be thought of as the molecular barcode that gives a readout of the epigenetic landscape of a given cellular population22,23 To date, the evaluation of CCSs in HD has remained unexplored. Given the central role of mHTT in the development of HD, we hypothesized that regulatory differences in genomic architecture at the HTT locus may exist between diseased individuals and healthy, unaffected controls.\n\nWe used EpiSwitch, an established proprietary industrial platform for monitoring CCSs, to assess chromatin architecture differences between pre-symptomatic and symptomatic HD patients and healthy, unaffected individuals. EpiSwitch readouts provide high resolution, reliable and high throughput detection of CCSs while simultaneously meeting the high bar of industry standards for quality control21. As such, this technology represents a powerful tool for screening, evaluation and monitoring of CCS in human disease24. This platform has been successfully utilized as a biomarker modality to stratify patients in the context of a variety of other diseases25–30, including as a non-invasive blood based biomarker for neurodegenerative conditions31,32.\n\n\nMethods\n\nAll blood samples were obtained from National BioService, LLC, a research biobank operating in compliance with the requirements of the International Society for Biological and Environmental Repositories. In total, 20 blood samples were used in this study; 10 healthy control (HC) samples (CAG repeats, n<35), and 10 HD samples (CAG repeats, n>39). For the HD samples, 7 were from symptomatic patients (HD-Sym) and 3 were from presymptomatic patients who had a diagnosis of HD but did not yet show any clinical symptoms (HD-Pre). One HD patient was taking tetrabenazine and one patient was taking sertraline. All samples were negative for human immunodeficiency virus, hepatitis B virus, hepatitis C virus and syphilis (Supplemental Table 1).\n\nWe wanted to identify chromosome conformations that differed between healthy controls (low CAG), presymptomatic HD patients (high CAG, no disease manifestation) and symptomatic HD patients (high CAG, disease manifestation). We focused on a ~225 kb region surrounding the HTT locus from (chr4: 3,033,588 to 3,258,170 as annotated in hg38) for our analysis. Using the CAG repeat expansion tract in exon 1 of HTT (chr4: 3,054,162 to 3,095,930) as the anchor point (“Anchor”), we defined five genomic zones surrounding the anchor to look at chromosome conformations that varied between sample groups (Figure 1 and Supplemental Table 2). These Zones were chosen based on: the presence of potential EpiSwitch anchoring sites, the presence of known disease-related SNPs (HD and other diseases), and the enrichment of known histone modification sites (H3K4me3, H3K36me3, and H3K27ac) in HD as found in the GWASdvV2 database (http://jjwanglab.org/gwasdb) (Figure 1).\n\nA visual overview of the genomic region investigated in this study. A ~225 kb region on chromosome 4 spanning the HTT locus was investigated. The anchor point (“Anchor” in track 4) was defined as a ~42 kb region spanning the CAG repeat tract in exon 1 of HTT (purple arrow at the top of the figure). We defined five Zones (Zones 1 -5 in track 4) based on overlap with EpiSwitch sites (track 3), SNPs related to Huntington’s disease (HD) (track 5) or other diseases (track 6), and observed methylation and acetylation (H3K4me3, H3K36me3 and H3K27Ac) differences between healthy control (HC) and HD (tracks 7 through 12).\n\nA search of the NCBI Gene Expression Omnibus database for previously reported HD epigenetic data was performed in February 201833. Peak-called ChIP-seq data for H3K4me3 from 12 (6 HD and 6 control samples) post-mortem prefontal cortex brain samples (bed format) was obtained (GSE68952)34. In addition, Bigwig tracks of ChIP-seq data for H3K27ac and H3K36me3 from HD iPSC-derived neural cell lines and control cell lines were also downloaded (GSE95342)35. The data tracks were loaded into the Integrative Genome Viewer (IGV)36 version 2.4 alongside the EpiSwitch and reference sequence annotations. Both visual and programmatic (BEDtools) comparisons were performed on the HTT locus to identify the five zones of interest.\n\nOxford BioDynamics proprietary EpiSwitch pattern recognition algorithm was used to identify high probability chromatin folding interactions with one “end” occurring in the anchor zone proximal to the CAG repeats and the other in any of the five zones of interest. A total of 61 interactions matched these criteria, and for practical reasons, 20 interactions were selected to cover interactions between the anchor site and all the zones of interest. Oxford BioDynamics automated primer design application was used to design oligonucleotide pairs that amplified the expected DNA sequence caused by the interaction when subjected to the chromosome conformation capture (3C) assay.\n\n3C and detection by PCR were performed as described previously25,28,30,37. Chromatin with intact chromosome conformations from 50 µl of blood sample from each patient sample was extracted using the EpiSwitch assay following the manufacturer's instructions (Oxford BioDynamics Plc). Quality control on all samples was done using the detection of a chromatin loop at the MMP1 locus, a historical internal control for 3C analysis30. Pooled 3C libraries for each of the sample types were generated to provide a generalized population sample for each of the C sample subgroups. Real-time PCR experiments were performed in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines38. Real-time PCR was performed with SYBR green with the CFX-96 (Bio-Rad) machine to identify the interactions with differing PCR product detection patterns between the sample types39. Oligonucleotides were tested on control templates to confirm that each primer set was working correctly. A full list of the primers and PCR conditions used in this study can be found in Supplementary File 1. The final nested PCR was performed on each sample in triplicates for the follow up data on individual patients with HD. This procedure permitted the detection of limited copy-number templates with higher accuracy. All PCR-amplified products were monitored on the LabChip® GX from Perkin Elmer, using the LabChip DNA 1K Version 2 kit (Perkin Elmer) and internal DNA markers were loaded on the DNA chip according to the manufacturer’s protocol using fluorescent dyes. Fluorescence was detected by laser and electropherogram read-outs translated into a simulated band on gel picture using the instrument software. The threshold of detection for the instrument was set by the manufacturer from 30 fluorescence units and above. All raw gel images for the PCR assays done in this study as well as a description of each set of comparisons can be found in Dataset 140.\n\nData analysis was performed in R (language and environment for statistical computing) version 3.5.1 (https://www.r-project.org)41. This included stats (version 3.6.0) and dplyr (version 0.7.6) packages for t-tests and R2 analysis & a ggplot2 (version 3.0)42 package for boxplots and regression plots.\n\n\nResults\n\nHC and HD samples were age (average 36.9 years for HC and 35.3 years for HD) and sex matched (10 male and 10 female), with the majority (70%) of HD cases being symptomatic (Table 1). All samples were from non-Hispanic or Latino whites. Average CAG repeats lengths were 25.7 for HC and 44.2 for HD (Table 1, Figure 2). There was no statistical difference in CAG repeat length between HD-Pre and HD-Sym (Figure 3). The average age at diagnosis for HD samples was 35.3 years and the average disease duration was 3.8 years with 7 out of 10 patients reporting symptoms of irritability, chorea, or both (Table 1).\n\nAge at Diagnosis was not available for 2 of the 10 HD patients. *Disease duration could not be calculated for 2 of the 10 HD patients. N/A, not applicable.\n\n(A) The healthy control (HC) (red) and Huntington’s disease (HD) (blue) samples showed no statistical difference in age. (B) The HC (red) patients showed no statistical difference in age from symptomatic HD (HD-Sym) (green). Pre-symptomatic HD (HD-Pre) patients were younger (average age = 25.3 years) than HD-Sym patients (average age = 39.6 years) (p = 0.02). There was a moderate negative relationship between disease duration and CAG repeat size (C) and a moderate positive relationship between age at diagnosis and CAG repeat size (D), though neither were statistically significant.\n\n(A) There was a statistically significant increase in CAG repeat length in Huntington’s disease (HD) patients (blue) relative to healthy control (HC) (red) (p = 1.08 E-7). (B) There was a statistically significant increase in CAG repeat length between HC (red) and Pre-symptomatic HD (HD-Pre) (blue) (p = 3.43 E-6) and symptomatic HD (HD-Sym) (green) (p = 9.50 E-8). There was no statistical difference in CAG repeat length between HD-Pre and HD-Sym (p = 0.09).\n\nOf the 20 interactions that were evaluated (Supplemental Table 3), we identified nine informative interactions. We identified two constitutive interactions and seven conditional interactions which were present in HD, but not healthy controls. Of the seven conditional interactions, three were present only in HD-Sym, and absent in HD-Pre.\n\nAll samples passed internal QC analysis for the MMP1 interaction (Supplementary Figure 1 and Supplementary Figure 2). Two constitutive (identified in all samples) chromatin loops were identified. Both loops were between the Anchor and Zone 2 with the first loop spanning 28 kb and the second loop spanning 34 kb (Figure 4).\n\nTwo constitutive interactions occurring in all patients (symptomatic Huntington’s disease (HD-Sym), Pre-symptomatic HD (HD-Pre) and healthy control (HC)) (red boxes) were observed in this study. Both interactions (C1 & C2) were between the anchor and zone 2 with C1 (A) spanning 28 kb and C2 (B) spanning 34 kb. TE=Tris-EDTA.\n\nWe identified seven conditional chromosome conformations that could discriminate between the different patient subgroups evaluated in this study. Specifically, we identified two chromosome interactions that were present in HC, but absent in all HD samples (Figure 5). The first interaction (I1) spanned the anchor and zone 4 and covered 77 kb while the second interaction (I2) spanned the anchor and zone 3 and covered 140 kb. We also identified two chromosome interactions that were present in HC and HD-Pre, but absent in HD-Sym samples. Both interactions (I3 and I4) spanned the anchor and zone 4, and covered 92 kb and 104 kb, respectively (Figure 6). Last, we identified three chromosome interactions that were present in HD-Sym samples, but absent in HD-Pre and HC samples. The first of these three conditional interactions (I5) spanned the anchor and zone 3, covering 122kb. Notably, this interaction included a SNP (rs362331) known to be a factor in the predisposition to develop HD. The second and third conditional interactions (I6 and I7) spanned the Anchor and Zone 1 and covered 185 kb and 174 kb, respectively (Figure 7). Last, we tested the absence or presence of all conditional interactions in individual HD samples. In the HD-Sym samples, we found the presence of at least one of the conditional markers (I5, I6 and I7) in six out of seven samples (Figure 8 and Supplementary Figure 3–Supplementary Figure 5). The odds ratios of each interaction being associated with symptomatic HD presentation were 30, 9 and 16 for I5, I6 and I7, respectively. A summary of all the interactions that were evaluated in this study are shown in Figure 9 and Supplementary Figure 6.\n\nTwo conditional interactions (red boxes) were observed in HC patients only and were absent from all Huntington’s disease (HD) patients. (A) The first interaction (I1) occurred between the Anchor and Zone 4 and spanned 77 kb. (B) The second interaction (I2) occurred between the Anchor and Zone 3 and spanned 140 kb. TE, Tris-EDTA.\n\nTwo conditional interactions (red boxes) were observed in healthy control (HC) patients and pre-symptomatic HD (HD-Pre) patients, but were absent from symptomatic HD (HD-Sym) patients. Both interactions (I3 & I4) occurred between the anchor and zone 4 and spanned 92 kb (A) and 104 kb (B), respectively. TE, Tris-EDTA.\n\nThree conditional interactions (red boxes) were observed in symptomatic HD (HD-Sym) patients only and were absent from HC and pre-symptomatic (HD-Pre) patients. (A) The first interaction (I5) occurred between the anchor and zone 3 and spanned 122 kb, including a HD-associated SNP known to be involved in disease progression. (B and C) The second and third interactions (I6 & I7) occurred between the anchor and zone 1, and spanned 185 kb and 174 kb, respectively. TE, Tris-EDTA.\n\nIn six out of seven individual HD-Sym samples, the presence of at least one of the three conditional interactions (I5, I6 and I7) was observed. I5, the interaction spanning the region that contains the rs362331 SNP, was observed in the greatest number of samples (5/7).\n\nOverview of the chromosomal conformation changes associated with the progression of HD. As patients progress from presymptomatic stages to symptomatic diseases, discrete, measurable and discriminating changes in the genomic architecture at the HTT locus are observed.\n\n\nDiscussion\n\nWhile it is well-known that individuals with greater than 39 CAG-repeats will get HD, the clinical onset of disease varies widely amongst individual patients and the factors that influence when the disease manifests clinically are less well characterized. Here we used EpiSwitch, an industrial platform for assessing chromatin architecture, to evaluate the epigenomic landscape of the HTT locus in HD patients and healthy, unaffected controls. We identified a set of seven interactions that when taken together as a CCS, could differentiate HD from unaffected controls and more importantly, could differentiate between presymptomatic and symptomatic HD patients. One of these interactions, specific for symptomatic HD, contains a SNP (rs362331) shown to be associated with a predisposing disease haplogroup. When taken together, these results provide an initial indication that a simple, non-invasive blood-based test evaluating a CCS can serve as a surrogate biomarker for assessing disease progression in HD.\n\nWhile it is known that the poly-Q repeat tract expansion and production of mHTT are the underlying causes of HD, the molecular events leading to the development of clinical symptoms are less well characterized. Several studies have looked at SNPs within the HTT locus as a potential contributor to disease onset. One recent SNP genotyping study of HD patients identified ~41 SNPs heterozygous in at least 30% of the patients, including the rs362331 C/T SNP in exon 50 of the HTT gene43. Perhaps more biologically relevant is that when the rs362331 SNP is allele-selectively knocked down using anti-sense oligonucleotides, siRNAs or miRNA, a dramatic reduction in the levels of mHTT protein is achieved both in vitro and in vivo, suggesting that this SNP and its surrounding genomic landscape play an important role in regulating mHTT levels44–46. In this study, we observed a chromosome conformation (I5) that was present in HD-Sym patients, was absent in HD-Asy and HCs, and overlapped with the rs362331 SNP. While requiring further study, this observation raises the interesting possibility that the production of neurotoxic mHTT in patients that have increased poly-Q tracts and a genetic predisposition to the early development of HD by the presence of the rs362331 SNP may be regulated at the level of higher-order chromatin structure. Another outstanding question in HD is how the disease is inherited in cases where neither parent has received a diagnosis. The two main prevailing hypotheses posit that 1) the carrier parent could have passed away from another factor before the onset of the disease and 2) “unstable” CAG repeat tracts expand with each generation. A third possibility also exists, in that at mid-range (35-50) repeats, individuals could be carriers without manifestation of the disease, but their progeny might be unable to compensate for the genetic defect through undefined mechanisms and will develop the disease. The HD patients evaluated in this study all had CAG repeats in this mid-range, raising the possibility that potential compensatory mechanisms in disease development may be mediated through differences in genomic architecture.\n\nHD is rare, in that there exists a simple test to definitively diagnose the disease, HTT gene sequencing and measurement of CAG repeat number. For clinical care and clinical trials, there are also several tests to measure disease severity, such as the Unified Huntington’s Disease Rating Scale, the Shoulson–Fahn Scale, and the Mini–Mental State Examination47–49. While these assessments measure different elements of an HD patients physical and mental well-being as a surrogate for disease severity, they are all subjective in nature and most are not specific for HD. What is missing are concrete molecular tools to monitor disease progression.\n\nAs of the time of this writing, there are 22 therapeutic agents for treating HD in different stages of preclinical and clinical development, half of which are in Phase 2 or Phase 3. Once further validated, the CCS reported here could be used in clinical trials as a surrogate outcome biomarker to assess the therapeutic efficacy of the drug in question. In addition to monitoring a symptomatic patient’s response to a particular therapy in clinical trials, another advantage of the approach described here lies in the information that can be obtained for pre-symptomatic patients. For most patients with HD, the pre-symptomatic period can last decades. Five of the seven (i3-i7) interactions identified here clearly separate presymptomatic HD patients from symptomatic ones, and when further validated could serve as an “early warning” indicator test for the onset of HD symptoms in presymptomatic carriers.\n\nThis study gives first evidence of detectable conditional differences in chromatin architecture specific for the manifestation of HD and correlated with known disease haplotypes. The major strength of this study lies in its unique approach, which is based on the latest developments in understanding the regulatory role of genomic architecture. While there have been several historical studies in HD aimed at developing disease progression biomarkers based on clinical, imaging and molecular measures50, to the best of our knowledge this is the first time that the assessment of higher-order chromatin structures in a clinically accessible biofluid has been applied in HD. With the successful application of EpiSwitch in another neurodegenerative condition, amyotrophic lateral sclerosis, as well as other non-neurological conditions such as melanoma, diffuse large B-cell lymphoma, chronic myelogenous leukemia, breast cancer, and rheumatoid arthritis, the results presented here further validate the use of regulatory conditional CCS as disease-related biomarkers25,28–31. A notable limitation of this study was the small sample size.\n\nWhile the data presented here offer a novel insight into the clinical progression of HD, this study was intended to be a proof-of-concept and not powered for statistical significance. A follow-up study using a larger patient cohort will be required for validation of these initial results.\n\n\nData availability\n\nDataset 1. Raw gel images for all PCR reactions performed in this study. Also included is a guide to the contents of the images. DOI: https://doi.org/10.5256/f1000research.15828.d22287340.", "appendix": "Grant information\n\nThis study was funded by Oxford BioDynamics, Plc.\n\n\nSupplementary material\n\nSupplementary Table 1. Clinical characteristics of the samples used in this study.\n\nClick here to access the data\n\nSupplementary Table 2. Genomic definitions for the Anchor point and Zones used in this study.\n\nClick here to access the data\n\nSupplementary Table 3. Summary of interactions tested in this study.\n\nClick here to access the data\n\nSupplementary Figure 1. Assessment of the MMP1 interaction for Quality Control.\n\nClick here to access the data\n\nSupplementary Figure 2. Assessment of the MMP1 interaction for Quality Control.\n\nClick here to access the data\n\nSupplementary Figure 3. Evaluation of the presence or absence of interaction 5 (I5) in individual samples from symptomatic HD (HD-Sym) patients.\n\nClick here to access the data\n\nSupplementary Figure 4. Evaluation of the presence or absence of interaction 6 (I6) in individual samples from symptomatic HD (HD-Sym) patients.\n\nClick here to access the data\n\nSupplementary Figure 5. Evaluation of the presence or absence of interaction 7 (I7) in individual samples from symptomatic HD (HD-Sym) patients.\n\nClick here to access the data\n\nSupplementary Figure 6. Summary of all interactions identified in this study.\n\nClick here to access the data\n\nSupplementary File 1. Primer sequences and PCR conditions used in this study.\n\nClick here to access the data\n\n\nReferences\n\nRoss CA, Aylward EH, Wild EJ, et al.: Huntington disease: natural history, biomarkers and prospects for therapeutics. Nat Rev Neurol. 2014; 10(4): 204–16. PubMed Abstract \| Publisher Full Text\n\nRawlins MD, Wexler NS, Wexler AR, et al.: The Prevalence of Huntington's Disease. Neuroepidemiology. 2016; 46(2): 144–53. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller JRC, Pfister EL, Liu W, et al.: Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells. Sci Rep. 2017; 7: 46740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPfister EL, Kennington L, Straubhaar J, et al.: Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington's disease patients. Curr Biol. 2009; 19(9): 774–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiniarikova J, Zanella I, Huseinovic A, et al.: Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease. Mol Ther Nucleic Acids. 2016; 5: e297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoussov K, Dolbeau G, Maison P, et al.: The unified Huntington's Disease Rating Scale for advanced patients: validation and follow-up study. Mov Disord. 2013; 28(14): 1995–2001. PubMed Abstract \| Publisher Full Text\n\nShoulson I, Fahn S: Huntington disease: clinical care and evaluation. Neurology. 1979; 29(1): 1–3. PubMed Abstract \| Publisher Full Text\n\nCockrell JR, Folstein MF: Mini-Mental State Examination. J Psychiatr Res. 1988.\n\nKilloran A, Biglan K: Biomarkers for Huntington’s disease: A brief overview. J Rare Dis Res Treat. 2016. Publisher Full Text" }	[ { "id": "43682", "date": "08 Feb 2019", "name": "Willeke M.C. van Roon", "expertise": [ "Reviewer Expertise Huntington disease", "gene expression analysis", "biomarker development", "therapy 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 investigates genome architecture differences at the HTT locus. There are several major issues with this study. The small sample size is the biggest issue. In my opinion it is too small to draw any conclusions. How a disease progression marker can be deducted from 3 presymptomatic samples (early disease state?) and 3 symptomatic samples is not clear. If the differences in chromosomal structure are indeed different around the HTT locus, what would be the functional consequence, and then this should be validated. Upon treatment, would this chromosomal then reverse? So far there is no evidence from brain tissue and fibroblast cells that there is a major difference in transcription from the wild type and mutant allele.\n\n“Although historically considered a monogenic disease, extensive research into the underlying pathology of HD suggests that the mechanisms leading to disease onset and progression are more complex than originally thought”. This is not a correct statement. It is a genetic disease; no polyQ expansion, no HD. But there are genetic modifiers that can influence onset and maybe progression.\n\n“HD is considered a paradigm of a disease characterized by epigenetic dysregulation”. This statement is not correct. HD is characterised by dysregulation in many cellular processes, and epigenetic deregulation is only one of them.\n\nThe number of samples is really too small. I have worked with human tissue for many years, and in my opinion these group sizes are too small. There are only 3 presymptomatic patients and 7 symptomatic. Furthermore, a CAG repeat range of 40-48 is not enough to say something about progression or severity. There is no appropriately age-matched control group for the presymptomatic HD group (Figure 1D).\n\n“Peak-called ChIP-seq data for H3K4me3 from 12 (6 HD and 6 control samples) post-mortem prefontal cortex brain samples (bed format) was obtained (GSE68952)34. In addition, Bigwig tracks of ChIP-seq data for H3K27ac and H3K36me3 from HD iPSC-derived neural cell lines and control cell lines\". So all the histone modification data that was used to identify zones of interest and to identify high probability folding interactions was based on data from neuronal cells/tissue. Is there anything known about how these histone modifications in brain relate to histone modifications in blood cells? I think this is a very important issue and potential pitfall of this paper.\n\nFor practical reasons, 20 interactions were selected from the identified 61 high probability chromatin interactions. Was this selection done randomly, for technical reasons, or were there any selection criteria? Please add this to the Methods section.\n\nFigure 2A is obsolete because the same samples are all used for Figure 2B. This figure can be removed from the paper. It is not very informative. All the relevant information is already in the table.\n\nFigure 3 illustrates that there are not enough samples in this study. The group size is much too small to include figures like this. Nothing is significant and the variability is large.\n\nFigure 4: The constitutive interactions in A is much weaker in the HD-Symp, group and in 4B much weaker in the HC group. How constitutive are these interactions? This Figure shows a summary. When I look at the individual data.\n\nFigure 8, the variability in the samples again is too large. What were the results for the individual presymp-HD?\n\nFigure 8 and 9: If I look at I6 for all the symp-HD samples, it was present in 3 and absent in 4, so in the majority it is absent. However in Figure 9 it is shown that interaction 6 is present in HD-symp.\n\nSince there is such a variation in absence and presence of interactions within the individual HD samples, there should also be a figure in the paper regarding the absence and presence of the interactions in the individual control samples. Also for the constitutive interactions.\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? 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? No", "responses": [ { "c_id": "4501", "date": "22 Mar 2019", "name": "Willem Westra", "role": "Author Response", "response": "We would like to thank Dr. van Roon for taking the time to read our paper in F1000Research and providing her thoughtful and insightful comments.We have included a point by point response to these comments in below:Comment #1: This paper investigates genome architecture differences at the HTT locus. There are several major issues with this study. The small sample size is the biggest issue. In my opinion it is too small to draw any conclusions. How a disease progression marker can be deducted from 3 pre-symptomatic samples (early disease state?) and 3 symptomatic samples is not clear. If the differences in chromosomal structure are indeed different around the HTT locus, what would be the functional consequence, and then this should be validated. Upon treatment, would this chromosomal then reverse? So far there is no evidence from brain tissue and fibroblast cells that there is a major difference in transcription from the wild type and mutant allele. Response: This aim of this study was not a full-scale analytical validation of biomarkers, but as stated in the Introduction, to introduce chromosome conformation signatures as a measurement modality with the potential to be developed into a clinical biomarker in the field of Huntington’s disease. It is known that current molecular modalities for assessing disease progression, including transcriptional profiling, have failed to provide clinicians with a reliable prognostic test for actionable stratification of patients with mid-range CAG extensions. This has been articulated by Prof. John Hardy, FRS, Chair of Molecular Biology of neurological Diseases, University College London, 2015 Breakthrough Prize, 2018 Brain Prize, at the London Lancet Neurology Conference in 2016. This study aimed to demonstrate, through an objective Odds Ratio evaluation on a small sized cohort, that a binary biomarker profile does show disseminating properties and therefore is worthy of further investigation in a larger cohort. The study used an approach that has been recently published in other disease areas, notably amyotrophic lateral sclerosis (ALS) and rheumatoid arthritis. In each of these studies, chromosome conformation signatures were developed and independently evaluated on an independent cohort of patients using a much larger sample size (Carini et al., J Translational Medicine 2018; Salter et al., EBioMedicine 2018). It is the intent to use the preliminary work described here to prompt further validation studies in a larger cohort of HD patients. Comment #2: “Although historically considered a monogenic disease, extensive research into the underlying pathology of HD suggests that the mechanisms leading to disease onset and progression are more complex than originally thought”.This is not a correct statement. It is a genetic disease; no polyQ expansion, no HD. But there are genetic modifiers that can influence onset and maybe progression. Response: We agree with the reviewer that from a basic standpoint, if there is no polyQ expansion, there is no HD. Our point in making this statement was that there are factors that influence disease onset and progression acting outside of polyQ expansion alone. This was recognized at the Lancet Neurology Conference in 2016 (see Response to Comment #1 above) and is supported by the observation that in HD individuals with mid-range polyQ expansions, there is a wide temporal range of clinical disease onset/manifestation and many patients remain asymptomatic for decades. In a similar vein, patients with identical polyQ expansions can manifest the disease at different times in their life. While there are several factors that may contribute here, epigenetic compensation is one of them and offers a novel molecular means to discriminate between genetic HD patients who manifest symptoms earlier in life and those who remain asymptomatic with delayed disease onset. This is a question of unmet clinical need and of high utility in clinical trials. Comment #3: “HD is considered a paradigm of a disease characterized by epigenetic dysregulation”.This statement is not correct. HD is characterised by dysregulation in many cellular processes, and epigenetic deregulation is only one of them. Response: Our statement acknowledges the role that changes in genome architecture, as an epigenetic modality, could play in clinical manifestation of HD, along with other regulatory processes. For clarity, we have adjusted the statement to meet the reviewer’s preferences. Comment #4: The number of samples is really too small. I have worked with human tissue for many years, and in my opinion these group sizes are too small. There are only 3 presymptomatic patients and 7 symptomatic. Furthermore, a CAG repeat range of 40-48 is not enough to say something about progression or severity. There is no appropriately age-matched control group for the presymptomatic HD group (Figure 1D). Response: As discussed, in a previous comment, we recognize the sample size as a limitation of the study and have noted this in the “Strengths and limitations” section. The primary goal of this study was to provide an initial proof-of-concept indication that the assessment of chromosome conformations at the HTT locus may be a useful molecular approach for assessing disease progression. We anticipate taking the chromosome conformations identified here as disseminating features into larger scale analytical validation studies, as has been done in previous studies (Carini et al., J Translational Medicine 2018; Salter et al., EBioMedicine 2018; Yan et al., Surgery 2019). Comment #5: “Peak-called ChIP-seq data for H3K4me3 from 12 (6 HD and 6 control samples) post-mortem prefontal cortex brain samples (bed format) was obtained (GSE68952)34. In addition, Bigwig tracks of ChIP-seq data for H3K27ac and H3K36me3 from HD iPSC-derived neural cell lines and control cell lines\".So all the histone modification data that was used to identify zones of interest and to identify high probability folding interactions was based on data from neuronal cells/tissue. Is there anything known about how these histone modifications in brain relate to histone modifications in blood cells? I think this is a very important issue and potential pitfall of this paper. Response: We direct the reviewer to a selection of peer reviewed studies in neurological and psychiatric disorders that clearly show significant epigenetic systemic profiling when measured in blood and compared to the primary sites of deregulation ( as an example, Lin et al. Characterization of Cross-Tissue genetic-epigenetic effects and their patterns… Genome Medicine 2018; Medrano-Fernandez and Barco, Nuclear Organization and 3D Chromatin Architecture in Cognition and Neuropsychiatric Disorders Molecular Brain 2016). The phenomenon of “horizontal transfer” and “exosome signalling” first demonstrated in oncological conditions (Feinberg, Key Role of Epigenetics in Human Disease Prevention and Mitigation New England J of Medicine 2018; Ratajczak Clinical and Translational Medicine 2016) underlies the extensive spectrum of published evidence for systemic epigenetic signatures. It is also important to note that the H3K27ac histone modification has been shown to correlate with changes in genome architecture (Huang et al. Genome Biology, 2016). Comment #6: For practical reasons, 20 interactions were selected from the identified 61 high probability chromatin interactions. Was this selection done randomly, for technical reasons, or were there any selection criteria? Please add this to the Methods section. Response: The 20 interactions were not selected randomly. The study design section and Fig.1 describes in detail the choices of regions and corresponding EpiSwitch sites (20 in total) for the analysis with the CAG repeat anchor site. This design and positions of the interacting sites is based on established methodology described recently in Carini et al. 2018; Salter et al., 2018, Yan et al., 2019). We are happy to further elaborate on the details of the design. Comment #7: Figure 2A is obsolete because the same samples are all used for Figure 2B. This figure can be removed from the paper. It is not very informative. All the relevant information is already in the table.Response: The rationale for including Figures 2A and 2B was to highlight that there was no statistical difference in CAG repeat length between symptomatic and presymptomatic HD patients. We agree that this information could be summarized in text and have updated Figure 2&3 into a combined Figure to reflect this. Comment #9: Figure 3 illustrates that there are not enough samples in this study. The group size is much too small to include figures like this. Nothing is significant and the variability is large.Response: We agree that and have combined Figures 2 and 3 to only include the main clinical comparisons. Comment #10: Figure 4: The constitutive interactions in A is much weaker in the HD-Symp, group and in 4B much weaker in the HC group. How constitutive are these interactions? This Figure shows a summary. When I look at the individual data. Response: A chromosome conformation is a binary modality, as described and defined in the literature (Crutchley et al. Chromatin Conformation signatures: ideal human biomarkers? Biomarkers Med 2010), and similar to the binary nature of a genetic variant. The presence of a conditional chromosomal conformation reflects active clonal support for systemic deregulation associated with a particular phenotype with copy numbers acting as a secondary input. This is not a continuous modality, like transcription, where for example a 20% increase/decrease in gene expression may correlate with a change in phenotype. Comment #11: Figure 8, the variability in the samples again is too large. What were the results for the individual presymp-HD? Figure 8 and 9: If I look at I6 for all the symp-HD samples, it was present in 3 and absent in 4, so in the majority it is absent. However, in Figure 9 it is shown that interaction 6 is present in HD-symp. Since there is such a variation in absence and presence of interactions within the individual HD samples, there should also be a figure in the paper regarding the absence and presence of the interactions in the individual control samples. Also for the constitutive interactions. Response: As described in the paper, for binary marker selection we first evaluated pooled samples with the same clinical annotation – HC, symp-HD, presymp-HD. With binary readouts on the absence/presence of individual interactions observed in these pooled samples, we only focused on markers that were conditional for specific group and absent across the others. For Odds Ratio calculations, individual samples were assessed for interactions that gave a positive (interaction present) readout in the pooled group. The assessment describer here provides a binary read out, and as such, the variability seen in continuous measurements is not applicable. As no interaction was observed at I5, I6 and I7 in pooled samples of presymptomatic patients or healthy controls (Figure 7) and assessment of these interactions in individual samples was not needed. Figure 9 is a representation of the absence/presence of interactions I1-I7 in pooled samples across the different groups. We hope the responses above have addressed the reviewer’s thoughts on the paper." } ] } ]	1	https://f1000research.com/articles/7-1757
https://f1000research.com/articles/8-653/v1	13 May 19	{ "type": "Research Article", "title": "Telomere length as a prognostic marker in colorectal cancer: a scoping review", "authors": [ "Gangmi Kim", "Kang Young Lee" ], "abstract": "Background: Telomeres are protective structures at both ends of a chromosome, which consist of repetitive DNA sequences (TTAGGG). Maintenance of telomere length is known to have important roles in carcinogenesis. However, there is no consensus about the prognostic role of telomere length in colorectal cancer. Methods: We conducted a scoping review using Pubmed and EMBASE as information sources through April 2019. Inclusion criteria were studies investigating telomere length and prognosis of colorectal cancer. Selected studies were reviewed to reevaluate the significance of telomere length in the prognosis of colorectal cancer. The aim of the study was to summarize the previous studies, to find consistent results, and to suggest future research. Results: In total, 12 studies were identified and 1955 patients were included from 2004 to 2019. Among 10 studies with tissue samples, two studies revealed better prognosis in patients with longer telomere length, and only stage IV patients were recruited in these two studies; four studies revealed better prognosis in patients with shorter telomere length or lower ratio of telomere length between cancer and normal tissue; four studies did not show any significant association between tumor length and prognosis. Two studies with blood samples presented contradictory results regarding the correlation between telomere length and survival rate. Conclusions: There was no consistent evidence to prove the prognostic value of telomere length in colorectal cancer. However, in a subgroup with the metastatic disease only, longer telomere length of tumor tissue was significantly associated with superior prognosis. Multicenter prospective studies with a large sample size are needed to verify the prognostic value of telomere length in colorectal cancer.", "keywords": [ "Telomere length", "colorectal cancer", "prognosis", "biomarkers" ], "content": "Introduction\n\nColorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Estimated number of newly diagnosed CRCs is 1,849,518 in 2018, according to the World Health Organization GLOBOCAN database. Moreover, CRC is the second-leading cause of cancer mortality worldwide. The number of deaths from CRC in 2018 is 880,792 according to the GLOBOCAN database. Clinical outcomes of CRC have been improved because of the introduction of regular screening, the advancement of surgical skills, and development of neoadjuvant therapy and adjuvant therapy1. However, the prognosis of stage IV CRC is poor, with fewer than 15% of patients surviving for 5 years2. Nowadays, personalized treatment is considered as a new strategy to improve clinical outcomes and effort is being made to explore appropriate biomarkers to identify patients who would respond to certain therapies1.\n\nAlthough numerous biomarkers have been studied in laboratories so far, only a couple of them are currently being used in practice. Telomere length might be considered as a potential biomarker to predict prognosis of CRC1. However, a limited amount of research has been conducted so far and there is a lack of solid evidence to support its prognostic role consistently3.\n\nTelomeres are protective structures at both ends of a chromosome. They consist of repetitive DNA sequences (TTAGGG) with associated proteins, and they protect the genome from degradation, recombination, and fusion4. Telomeres shorten with each cell division by loss of DNA termini, and finally, cells undergo senescence or apoptosis following activation of the DNA damage response, when telomeres become too short to protect chromosome ends after repeated cell divisions. However, cancer cells overcome senescence or apoptosis and acquire immortality by maintaining telomere length. There are two mechanisms for telomere maintenance: 1) transcriptional activation of telomerase and 2) activation of alternative lengthening of telomeres (ALT). Telomerase is expressed in 85–95% of all cancer cells and the ALT pathway is activated in 5–15%5.\n\nMaintenance of telomere length is considered to have important roles in carcinogenesis and researchers studies about the role of telomere length as a prognostic marker in CRC. However, conflicting results have been published and there is currently no consensus. Thus, this study aimed to review all the published studies including the newest one and to evaluate the significance of telomere length as a prognostic marker for CRC.\n\n\nMethods\n\nPubmed and Embase were systemically searched to identify studies investigating telomere length and CRC prognosis in adherence with the Preferred Reporting Items for Systematic review and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) checklist6. MeSH terms including “telomere”, “colorectal neoplasms”, “colonic neoplasms”, and “rectal neoplasms” were used for Pubmed search. Emtree terms including “telomere length”, “colorectal cancer”, “colon cancer”, and “rectum cancer” were used for Embase search. Studies were selected for review if they met all of the following criteria: 1) human CRC cancer patients were included; 2) telomere length from a tissue sample or from blood sample was measured; 3) clinical outcomes were reported as survival rate or disease relapse. The latest search was performed on April 22, 2019. No date limits were imposed and there was no language limitation.\n\nTwo researchers (G.K. and K.Y.L.) reviewed all included studies and extracted the following data independently: author, year, country, origin of samples, cancer site, sample size, mean (or median) age of involved patients, tumor stage, assay or detection method for telomere length, follow up period, and survival outcome. A pilot test for three articles to check the data extraction form and to ensure consistency was performed before data extraction.\n\nA detailed review protocol is available as Extended data7.\n\n\nResults\n\nIn total, 533 studies were initially screened from Pubmed database and 815 studies were screened from Embase. Among 1,348 studies, 1,336 studies were excluded and 12 studies were finally identified to report the association between telomere length and prognosis of CRC between 2004 and 2019. The included cases from 12 studies were 1,955. Of these studies, ten measured telomere length from tissue samples (Table 1) and two measured telomere length from peripheral leukocytes (Table 2). There were seven studies from Europe, three from Asia, one from the USA, and one from Australia.\n\nOS, overall survival rate; DFS, disease-free survival rate; TL, telomere length; TL ratio, ratio of telomere length between cancer and normal tissue.\n\nOS, overall surival rate; DFS, disease-free survival rate; TL, telomere length.\n\nIn brief, the results from 12 studies are contradictory. First, two of ten studies analyzing tissue samples showed better prognosis of patients with longer telomere length, especially in patients with metastasis8,9.\n\nBalc’h et al. measured telomere length from 125 CRC tissue samples8. A significant shortening of telomere length in the tumor tissue compared with the normal tissue was observed. They reported that the overall survival rate was significantly correlated with telomere length in metastatic disease (n=28, p=0.03). In metastatic disease, patients with longer telomeres survived longer than those with shortened telomeres. However, there was no statistically significant difference in overall survival rate between the maintained telomere length group and shortened telomere length group when analyzing localized disease group (n=97) or the entire group (n=125). In addition, they observed that telomere length was significantly associated with the occurrence of mutation in KRAS. They found that the shorter the telomeres in healthy tissue were, the longer the telomeres in tumor tissue were. They suggested that telomere length in healthy normal tissue might influence telomere maintenance mechanisms in a tumor.\n\nAugustine et al. measured telomere length from 75 CRC tissue samples and explored the efficacy of telomere length as a predictor for response to anti-TGFR therapy in metastatic disease9. They first measured telomere length in 21 human-derived CRC cell lines. When the cell lines were treated with cetuximab, a monoclonal antibody to EGFR, growth of cell lines with a longer telomere length was inhibited to a significantly larger degree than cell lines with a shorter telomere length (p=0.02). When the analysis was limited to K-ras wild-type cell lines only, growth of cells with a longer telomere length was more inhibited by cetuximab treatment compared with cells with a shorter telomere length (p=0.04), which was similar to above. Next, they analyzed telomere length in patients’ tumor samples. In the analysis of all human CRC samples, progression-free survival after anti-EGFR therapy was significantly longer in patients with longer telomere length compared with that of patients with shorter telomere length (n=75, p=0.026). Furthermore, when the analysis was narrowed down to the patients with K-ras wild-type tumors only, again, patients with longer telomere length showed longer progression-free survival than patients with shorter telomere length (n=43, p=0.012). The authors suggested that telomere length could play a role as a marker for predicting the benefit of anti-EGFR treatment in metastatic CRC.\n\nThe two aforementioned studies suggested the telomere length as a prognostic marker in metastatic CRC8,9. The longer the telomere, the better the prognosis was. On the contrary, the next four studies among the ten to examine tissue samples showed better prognosis of patients with shorter telomere length10–13.\n\nFernández-Marcelo et al. analyzed 132 CRC tissue samples and corresponding noncancerous normal tissue samples, and measured telomere length, a ratio of telomere length in cancer to normal tissue (TL ratio), and telomerase activity10. Patients with shorter telomere length showed a significantly longer disease-free survival compared with those with longer telomere length (p<0.001). Moreover, patients with the lowest TL ratio never experienced recurrence during the follow-up period (median 60 months, p=0.043). Cox multivariate analysis also showed that mean telomere length was an independent prognostic factor for disease-free survival (p=0.017). The authors suggested the use of telomere status as an independent prognostic factor.\n\nValls et al. analyzed 147 CRC tissue samples and paired normal tissue samples. The authors measured the telomere length of the samples and calculated the TL ratio, which is defined as a ratio of telomere length in cancer to normal tissue11. Univariate analysis and multivariate Cox regression analysis both showed a significant relationship between TL ratio and overall survival rate. Patients with lower TL ratios exhibited a significantly longer overall survival rate (n=125, p=0.014). The TL ratio was an independent prognostic factor for overall survival in CRC. However, there was no significant correlation between TL ratio and the disease-free survival rate.\n\nGarcia-Aranda et al. explored 91 CRC tissue samples12. They measured telomere length in CRC tissue samples and paired normal tissue samples. They also accessed telomerase activity and expression level of telomeric repeat-binding factor (TRF1), which is one of six subunits of the shelterin complex. Patients with longer telomeres showed a significantly shorter disease-free survival rate (p=0.02). Cox regression analysis also proved that telomere length was an independent prognostic factor (p=0.04). In patients with Dukes stage C tumors, both longer telomere length and higher TL ratio were significantly associated with shorter disease-free survival rate (p=0.03 and p=0.04, respectively). Additionally, patients with shortened telomeres expressed higher TRF1 levels than patients without telomere shortening. Furthermore, among patients with telomerase-positive tumors, lower TL ratio and TRF1 over-expression was associated with longer disease-free survival (p=0.03 and p=0.05, respectively).\n\nGetler et al. analyzed telomere length and hTERT expression in 57 CRC tissue samples and adjacent normal tissue samples13. Patients with longer TL ratio showed a significantly shorter overall survival rate compared with patients with shorter TL ratio (p<0.002). In multivariate Cox regression analysis, TL ratio was an independent prognostic factor; longer TL ratio predicted shorter overall survival (p<0.03). Additionally, telomere length and hTERT expression correlated significantly in cancer tissues and normal tissues (p<0.001 and p<0.001, respectively), and telomere length was shorter in cancer tissue compared with normal mucosa (p<0.001). The authors suggested that hTERT-mediated telomere stabilization might play a role in the progression and prognosis of CRC.\n\nThe following four studies did not prove an association between telomere length and prognosis of CRC14,15–17. Bae et al. analyzed telomere length in 60 CRC tissue samples and corresponding nonmalignant normal tissue samples14. In the survival analysis, there was no association between telomere length and overall survival rate or disease-free survival rate. They also evaluated the expression level of telomeric repeat-containing RNA (TERRA) in cancer tissue and normal tissue. TERRA refers to a class of long noncoding RNAs which forms an integral component of telomeric heterochromatin20. TERRA is also known to regulate telomere length and telomerase activity21. The analysis showed a significant association between TERRA expression and telomere length (p<0.05). However, it did not prove any statistical significance of TERRA as a prognostic factor for overall survival or disease-free survival although there was a tendency that survival was better in TERRA high group.\n\nSuraweera et al. analyzed telomere length in 419 CRC tissues and adjacent normal tissues15. Telomere length was significantly shorter in cancer tissues than in normal tissues (p<0.001). Survival analysis was performed only in stage II and III CRC (n=281). There was no significant association between overall survival (n=281) or disease-free survival (n=246) and tumor length or TL ratio.\n\nLopez-Doriga et al. analyzed telomere length in cancer tissues and adjacent normal tissues from 42 patients with stage II colon cancer16. Significant shortening of telomere length was identified in cancer tissues as compared to their adjacent normal tissues (p<0.01). No significant relationship was observed between the recurrence rate and telomere length or TL ratio.\n\nKojima et al. measured telomere length, length of telomere 3’-overhang (3’-OH) and telomerase activity from 106 CRC tissue samples and corresponding noncancerous normal mucosa17. There was no significant association between telomere length and prognosis. However, patients with shortened 3’-OH showed a significantly increased survival rate compared with those without 3’-OH shortening among patients with telomerase-activated cancers, (p=0.018). Additionally, expression levels of telomere binding proteins (TBPs) were analyzed in cancer samples and normal mucosa samples. The analysis revealed all TBPs, except for protection of telomeres 1 (POT1), which is one of six subunits of the shelterin complex, were downregulated in cancers. In the telomerase-activated cancers, there was a significant correlation between the length of 3’-OH and expression level of POT1. The authors suggested that elongation of telomeric 3’-OH could increase malignant potential in CRC and that might be regulated by POT1.\n\nLastly, there were two studies analyzing telomere length in peripheral leukocytes from CRC patients18,19. The two studies presented conflicting results. Chet et al. analyzed telomere length of peripheral leukocytes from 571 CRC patients, who were followed up for 28 months18. Patients with longer telomere length presented significantly longer overall survival and longer disease-free survival (p<0.001 and p<0.001).\n\nSvenson et al. analyzed 130 blood samples of CRC patients and followed up the patients for 202 months19. On the contrary to the previous study, patients with shorter telomere length showed significantly longer overall survival (p=0.030).\n\n\nDiscussion\n\nThis study reviewed the data of 1,955 CRC patients from 12 publications. Survival outcome according to the telomere length was the primary endpoint. Before this study, there were two review articles to evaluate the prognostic value of telomere length for CRC22,23. All the literature analyzed by these two articles was included for review in our study.\n\nIn 2016, Jia et al. reviewed five studies and performed meta-analysis22. Among the five studies initially included in the review, two studies were excluded because Chen’s study used blood samples to measure telomere length18 and Kojima’s study did not report hazard ratios and 95% confidence intervals17. Thus, only three studies with 279 patients were meta-analyzed finally. The result was that longer telomere length was significantly associated with poorer overall survival (HR=2.70, 95% 1.51 to 4.84, p=0.001) and the authors concluded that some evidence exists for telomere length as a prognostic factor for overall survival in CRC.\n\nIn 2017, Wang et al. reviewed eight studies and performed a meta-analysis to investigate the association between telomere length and prognosis of CRC23. Two of the studies used blood samples as materials18,19, unlike the others which used tissue samples; however, the pooled analysis included all the eight studies. As a result, there was no significant association between telomere length and survival for CRC patients.\n\nAlthough Jia et al. concluded telomere length had a prognostic value for overall survival in CRC patients, only three studies were included in the meta-analysis and consequently, high risk of bias could not be avoided. And their study did not assess the association between telomere length and disease-free survival22. Following Jia’s study, Wang et al. performed meta-analysis with eight studies only to fail to prove any association between telomere length and survival outcome23.\n\nTherefore, there has been no solid evidence to confirm the role of telomere length as a prognostic marker in CRC so far. Three more studies were published after Wang’s study and we planned a new literature review to reevaluate the significance of telomere length in CRC. The aim of our study was to summarize the results of previous studies; to find consistency from the previous studies; to aid further research by analyzing the strengths and weaknesses of the previous studies.\n\nIn our review, as summarized in Table 1 and Table 2, the studies were categorized according to the sample material. The majority of the included studies used cancer specimen for measuring telomere length and two studies used blood sample of patients. In the first category with tissue samples, the results varied. Two studies revealed better prognosis in patients with longer telomere length, and only stage IV patients were recruited in these two studies8,9; four studies revealed better prognosis in patients with shorter telomere length or lower TL ratio10–13; four studies did not show any significant association between tumor length and prognosis14,15–17. In the next category with blood samples, two studies presented contradictory results regarding the correlation between telomere length and survival rate18,19.\n\nIn conclusion, we found no consistency in the literature. There was no consistent evidence to prove the prognostic value of telomere length in colorectal cancer. However, in a subgroup with the metastatic disease only, longer telomere length of tumor tissue was significantly associated with superior prognosis.\n\nMore evidence is needed to support the significance of telomere length in the prognosis of CRC. To avoid the risk of bias, multicenter prospective studies with a large number of patients are warranted. Additionally, presenting data as being categorized by certain clinical characteristics such as TNM stages, tumor locations, or genders is encouraged to facilitate subgroup analyses. And finally, providing original data with statistics could be a support for future research even if study results are insignificant or discouraging.\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: review protocol.docx. https://doi.org/10.6084/m9.figshare.8075207.v17.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nPRISMA-ScR checklist for article “Telomere length as a prognostic marker in colorectal cancer: a scoping review”. https://doi.org/10.6084/m9.figshare.8072540.v16.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBisoffi M, Heaphy CM, Griffith JK: Telomeres: prognostic markers for solid tumors. Int J Cancer. 2006; 119(10): 2255–2260. PubMed Abstract \| Publisher Full Text\n\nArmaghany T, Wilson JD, Chu Q, et al.: Genetic alterations in colorectal cancer. Gastrointest Cancer Res. 2012; 5: 19–27. PubMed Abstract \| Free Full Text\n\nBertorelle R, Rampazzo E, Pucciarelli S, et al.: Telomers, telomerase and colorectal cancer. World J Gastroenterol. 2014; 20(8): 1940–1950. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith L, Luchini C, Demurtas J, et al.: Telomere length and health outcomes: An umbrella review of systematic reviews and meta-analyses of observational studies. Ageing Res Rev. 2019; 51: 1–10. PubMed Abstract \| Publisher Full Text\n\nOkamoto K, Seimiya H: Revisiting Telomere Shortening in Cancer. Cells. 2019; 8(2): pii: E107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim G: PRISMA-ScR checklist.doc. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8072540.v1\n\nKim G: review protocol.docx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8075207.v1\n\nBalc’h EL, Grandin N, Demattei MV, et al.: Measurement of Telomere Length in Colorectal Cancers for Improved Molecular Diagnosis. Int J Mol Sci. 2017; 18(9): pii: E1871. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAugustine TA, Baig M, Sood A, et al.: Telomere length is a novel predictive biomarker of sensitivity to anti-EGFR therapy in metastatic colorectal cancer. Br J Cancer. 2015; 112(2): 313–318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernandez-Marcelo T, Sánchez-Pernaute A, Pascua I, et al.: Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer. PLoS One. 2016; 11(2): e0149626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValls C, Piñol C, Reñé JM, et al.: Telomere length is a prognostic factor for overall survival in colorectal cancer. Colorectal Dis. 2011; 13(11): 1265–1272. PubMed Abstract \| Publisher Full Text\n\nGarcia-Aranda C, de Juan C, Diaz-Lopez A, et al.: Correlations of telomere length, telomerase activity, and telomeric-repeat binding factor 1 expression in colorectal carcinoma. Cancer. 2006; 106(3): 541–551. PubMed Abstract \| Publisher Full Text\n\nGertler R, Rosenberg R, Stricker D, et al.: Telomere length and human telomerase reverse transcriptase expression as markers for progression and prognosis of colorectal carcinoma. J Clin Oncol. 2004; 22(10): 1807–1814. PubMed Abstract \| Publisher Full Text\n\nBae SU, Park WJ, Jeong WK, et al.: Prognostic impact of telomeric repeat-containing RNA expression on long-term oncologic outcomes in colorectal cancer. Medicine (Baltimore). 2019; 98(14): e14932. PubMed Abstract \| Publisher Full Text\n\nSuraweera N, Mouradov D, Li S, et al.: Relative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer. Oncotarget. 2016; 7(24): 36474–36488. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopez-Doriga A, Valle L, Alonso MH, et al.: Telomere length alterations in microsatellite stable colorectal cancer and association with the immune response. Biochim Biophy Acta Mol Basis Dis. 2018; 1864(9 Pt B): 2992–3000. PubMed Abstract \| Publisher Full Text\n\nKojima K, Hiyama E, Otani K, et al.: Telomerase activation without shortening of telomeric 3’-overhang is a poor prognostic factor in human colorectal cancer. Cancer Sci. 2011; 102(2): 330–335. PubMed Abstract \| Publisher Full Text\n\nChen Y, Qu F, He X, et al.: Short leukocyte telomere length predicts poor prognosis and indicates altered immune functions in colorectal cancer patients. Ann Oncol. 2014; 25(4): 869–876. PubMed Abstract \| Publisher Full Text\n\nSvenson U, Öberg Å, Stenling R, et al.: Telomere length in peripheral leukocytes is associated with immune cell tumor infiltration and prognosis in colorectal cancer patients. Tumor Biol. 2016; 37(8): 10877–10882. PubMed Abstract \| Publisher Full Text\n\nLuke B, Lingner J: TERRA: telomeric repeat-containing RNA. EMBO J. 2009; 28(17): 2503–2510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Zhao L, Lu S: Role of TERRA in the regulation of telomere length. Int J Biol Sci. 2015; 11(3): 316–323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJia H, Wang Z: Telomere Length as a Prognostic Factor for Overall Survival in Colorectal Cancer Patients. Cell Physiol Biochem. 2016; 38(1): 122–128. PubMed Abstract \| Publisher Full Text\n\nWang W, Zheng L, Zhou N: Meta-analysis of associations between telomere length and colorectal cancer survival from observational studies. Oncotarget. 2017; 8(37): 62500–62507. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "54187", "date": "07 Oct 2019", "name": "Laura Reyes-Uribe", "expertise": [ "Reviewer Expertise Molecular epidemiology of colorectal cancer", "colorectal cancer and premalignant tumor immunology", "hereditary colorectal cancer" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study aims to summarize the evidence regarding the role of telomere length as a prognostic biomarker in colorectal cancer. It is a systematic, non-quantitative review of the literature, that concludes that there is inconsistent evidence regarding the utility of this biomarker for predicting survival. The paper provides very helpful summary tables, describing pertinent clinical information about the patients included in individual studies, along with the methods used to measure telomere length or ratio. Below are some comments that we think could strengthen this manuscript and the discussion regarding these biomarkers.\n\nGeneral comments\nAt least three biomarkers are reviewed: 1) tumor telomere absolute length, 2) tumor vs non-tumor telomere length ratio and 3) peripheral leukocyte absolute telomere length. Age-adjustment and additional variables (e.g. telomere 3’-overhang) are also included in some of the studies reviewed. Although correlations between these metrics may exist 1, we think each one should be discussed and evaluated separately, unless evidence to justify their grouping is presented.\nThe review concludes that “multicenter prospective studies with large numbers of participants are warranted”. Such studies may be warranted if these biomarkers showed consistent or aggregated independent prognostic value; had been suggested to improve current clinical prognostication methods (e.g. the Immunoscore) or have therapeutic relevance (e.g. MSI); and could be prospectively measured and interpreted (e.g. values can be prospectively classified as good or poor prognosis). The data presented does not seem to suggest that any of the three main telomere-related biomarkers meet these criteria.\nIt would also be helpful to briefly discuss why a quantitative meta-analysis of the different telomere-related biomarkers was not performed. It seems like the heterogeneity of the studies would not lend itself well to such an analysis, and could lead to inaccurate conclusions; this is worthwhile mentioning.\nIntroduction\nThe introduction could benefit from discussion of telomere length across the spectrum of colon premalignancy to metastatic disease 2,3. It would also be helpful to briefly summarize the data regarding the association between telomere length and other prognostic and molecular features of colorectal cancer (e.g. stage, MSI status, sidedness, KRAS/BRAF), as well as how these associations may impact the independent prognostic value of telomere length. For example, shorter telomere length has been associated with early-stage tumors, right-sided tumors and microsatellite unstable (MSI-H) tumors in some studies 2,3.\n\nMinor comment: references 1 and 2 do not seem to be supporting the sentences preceding them. 1 is referring to improved outcomes in CRC but the reference is about telomeres and 2 is regarding outcomes in stage IV disease but the reference is about genetic alterations.\n\nMethods\n\nMinor comment: if the authors update their publication, they could consider including another paper that has since been published 2.\n\nResults\n\nThe study by Suraweera et al. is the largest study in the review, and seemingly the most comprehensive and rigorous. It would be helpful to discuss this study more in detail, including its use of age-adjusted variables.\nIt is worth noting that Lopez-Doriga et al. did not directly measure telomere length. Rather, their length and ratio estimates are based on whole-exome sequencing data in MSS tumors. They found an association with somatic mutation rate and with gene expression of several immune-related pathways that is also worth noting4.\nMinor comment: the PRISMA flow diagram5 or a slightly more detailed description of the study inclusion/exclusion process would be helpful to include. It would be unusual to have 1,348 unique studies with no overlap between PubMed and Embase, for example.\nMinor comment: there is a typo mentioning anti-TGFR instead of anti-EGFR.\n\nDiscussion\nFocusing the discussion on the current literature review, its limitations and the reliability of the results may be more informative than an extensive discussion of previous meta-analyses (these could be briefly summarized).\nThe authors note that Wang et al. included very different (tumor and blood-based) studies in their meta-analysis. As mentioned above, unless the authors provide evidence to the contrary, these should likely not be grouped together and thus the conclusions in the paper by Wang et al. seem to not be applicable. The following sentences in the discussion do not seem to reflect this major limitation of the meta-analysis of Wang et al: “As a result, there was no significant association between telomere length and survival for CRC patients.” and “Wang et al. performed meta-analysis with eight studies only to fail to prove any association between telomere length and survival outcome”.\nIt would be helpful to discuss the associations between telomere length and clinical/molecular characteristics found in several studies, and how these can potentially confound the study of telomeres as prognostic biomarkers. Given the importance of the immune response to colorectal cancer in predicting survival 6-8, it may be worthwhile to summarize any data regarding the relationship between telomere length and the immune response to colorectal cancer.\n\nIf possible, it would be helpful to suggest which of the telomere-related biomarkers is the best candidate for further studies (based on CRC studies or studies in other cancers). If none of the markers is preferable, we would mention that as one of the challenges in validating the role of telomere-related biomarkers.\nThe conclusion regarding patients with metastatic disease (in the discussion and abstract) is based on a subgroup analysis of 28 patients in the study by Balc’h et al. and another study where all patients were treated with anti-EGFR therapy. We would be hesitant to draw such a strong conclusion based on these data.\nMinor comment: The sentence “This study reviewed the data of 1,955 CRC patients from 12 publications” may be misinterpreted as having performed a patient-level analysis as opposed to aggregated data analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "61326", "date": "19 Mar 2020", "name": "Rajiv Kumar", "expertise": [ "Reviewer Expertise Cancer genetics with specific interest in genetics of telomerases and telomeres." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 described in the manuscript is based on data available for association between telomere-length and the disease prognosis in patients with colorectal cancer. The data presented are curated from published studies on the topic.\n\nGeneral comments: The idea of the study is to ascertain whether telomere length can be a biomarker of the disease outcome in colorectal cancer. The studies reviewed include those that have measured telomere length in tumor tissues, tumor tissues/adjacent normal tissues or in peripheral leukocytes. The methodology used in the studies included ranged from qPCR to Sothern blotting. And outcomes in those studies, not surprisingly, are not concordant. The reasons for that are multi-fold. One of the prime reasons can be the lack of enough statistical power, which hampers many such studies. The other reasons could be methodological or quality of tissues screened. All those issues should be part of consideration for the discussion in this study.\nSuggestions: I think in a study of this scope, it is important to provide a context for the potential of telomeres as biomarkers of cancer risk and as outcome predictors. There have been several studies, which have shown association of leukocyte telomere length with increased risk of several cancers1. In a majority of cancers, the increased risk has been associated with longer telomeres and in some cancers with shorter telomeres1,2. And many later studies have used Mendelian randomization to provide genetic basis for such association as barring some exogenous factors, the telomere length is genetically determined1,3. Similarly, several studies have shown that shorter leukocyte telomeres associate with poor outcome in different cancers4. It will give the paper an overall uplift and provide readers the necessary context. And accordingly, the results can be discussed within that context and provide a space to highlight the shortcomings in the studies included in the analysis.\nIt is also pertinent to discuss the methodology. Most of the studies included in the analysis have used quantitative PCR that actually measures a ratio between telomere content and the albumin gene content in a given DNA sample5. That method itself has several versions that could impact the results. Two studies have use Southern blot that actually gives the measure of telomere length in kilo-bases.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-653
https://f1000research.com/articles/8-652/v1	13 May 19	{ "type": "Case Report", "title": "Case Report: Schwannoma of the sigmoid colon: a case report of a rare colonic neoplasm and review of literature", "authors": [ "Gangmi Kim", "Sun Il Kim", "Kang Young Lee", "Sun Il Kim" ], "abstract": "Background: Schwannomas are tumors originating in Schwann cells of the peripheral nerve system and uncommonly develop in the gastrointestinal tract. Sigmoid colon schwannomas are very rare and only 28 cases have been reported. This study aims to report a case of a sigmoid colon schwannoma and present a literature review. Case report: We report a case of a 66-year-old female with asymptomatic sigmoid colon schwannoma. The patient underwent a screening colonoscopy and about 4cm sized submucosal tumor was identified at the sigmoid colon. A colonoscopic biopsy was performed and the microscopic exam revealed an ulcerated lesion with a proliferation of fibroblast-like spindle cells beneath ulcer, which was insufficient for diagnosis. Abdominopelvic computerized tomography (CT) scan showed a well-defined, well-enhancing, round shaped and slightly heterogenous mass at the sigmoid colon. No distant metastasis was identified in abdominopelvic CT and chest CT scans. Carcinoembryonic antigen level was within a normal range (1.33ng/mL). The patient underwent laparoscopic anterior resection. Immunohistochemical staining of the resected specimen showed positivity for S-100 protein in tumor cells and schwannoma was diagnosed post-surgically. Surgical resection margins were free from tumor and no regional lymph node metastasis was reported. Conclusion: Colon schwannomas are rare diseases. Most cases of colon schwannomas are accidentally identified during screening colonoscopy. The tumors usually present as submucosal masses and colonoscopic biopsies are mostly non-diagnostic. Surgical resection is required, and definitive diagnosis is made by confirming S-100 positive tumor cells in immunohistochemical analysis. Most cases are benign; a few cases have been reported to be malignant. Surgical resection with free negative margins is the treatment of choice", "keywords": [ "Colon", "schwannoma", "colonic neoplasms", "colectomy" ], "content": "Introduction\n\nSchwannomas are a common type of tumor of peripheral nerve in adults which originate in Schwann cells. These tumors mainly present along the peripheral nerves and are rarely identified in the gastrointestinal (GI) tract1,2. GI tract schwannomas develop most frequently in the stomach (83%), and less frequently in the small intestine (12%), colon and rectum3. Sigmoid colon schwannomas are very rare and only 28 cases have been reported so far3,4. Most colon schwannomas are incidentally identified as submucosal tumors on screening colonoscopy1,5. Colonoscopic biopsies alone usually provides limited information and definite diagnosis is made after surgical resection1. Most of the colon schwannomas are benign and surgical resection with adequate free resection margins is the treatment of choice6. Here we present a case of sigmoid colon schwannoma and discuss the clinical features of the disease with a literature review.\n\n\nCase report\n\nA 66-year-old Asian female patient, who was a housewife, visited a local clinic for a routine screening colonoscopy in mid-January 2018. During the colonoscopy, a submucosal tumor sized about 4cm was identified at the sigmoid colon (Figure 1) and biopsy was performed. The microscopic exam of the biopsied specimen showed an ulcerated lesion with a proliferation of fibroblast-like spindle cells beneath the ulcer, which was insufficient for a definite diagnosis.\n\nAbout 4cm sized submucosal tumor was identified at the sigmoid colon.\n\nThe patient was referred to our hospital at the end of January 2018. She presented no specific symptom and physical examination showed no specific finding. She had a history of hypertension and a benign breast mass. She had a positive family history of cancer: her father had gastric cancer, and her uncle had lung cancer.\n\nAn abdominopelvic computerized tomography (CT) scan revealed 4.4cm sized well-circumscribed, well-enhanced, round-shaped mass in the sigmoid colon, which was slightly heterogenous inside. No intraabdominal metastasis was identified (Figure 2A–2B). Chest CT scan showed no intrathoracic metastasis. Carcinoembryonic antigen (CEA) level was 1.33ng/mL, which was within a normal range (0.0–5.0ng/mL). Other laboratory test results were also within normal ranges. Differential diagnosis was 1) gastrointestinal stromal tumor (GIST); and 2) neuroendocrine tumor (NET).\n\nA well-circumscribed, well-enhanced, round-shaped mass was identified at the sigmoid colon. (A) Coronal view. (B) Axial view.\n\nThe patient underwent a laparoscopic anterior resection. On laparoscopic exploration, an extruding mass was identified at the anterior wall of the sigmoid colon and no metastasis was observed. The sigmoid colon was mobilized and the inferior mesenteric artery was low ligated. Sigmoid colon resection with end-to-end anastomosis was performed.\n\nOn examining the resected specimen, about 4.5 × 4.0cm sized round mass was observed on the surface of the serosa and there was no tumor infiltration to the serosa (Figure 3A–3B). The tumor was located 7cm from the proximal resection margin and 4cm from the distal resection margin. On sections after fixation, the cut surface showed a yellowish mass (4.2×3.2cm), which was abutting on the circumferential resection margin. The mass was relatively well-demarcated without encapsulation (Figure 4). On hematoxylin and eosin (H&E) stain, the tumor was composed of spindle cells with low nuclear atypia, with nuclear palisading growth pattern, and lymphoid cuffing surrounding tumor cells were identified (Figure 5A–5C). Mitosis was rarely observed (1/50 in high-power field). The remaining mucosa and serosa were grossly unremarkable. The resection margins were free from tumor. Lymph node metastasis was zero in 13 regional lymph nodes. On immunohistochemical analysis, s-100 was strongly positive in tumor cells; otherwise, c-kit, CD34, and SMA were negative (Figure 6A–6D). Finally, the diagnosis was a benign schwannoma of the sigmoid colon.\n\n(A) 4.5 × 4.0cm sized round, protruding mass was observed on the surface of the serosa. (B) The photo was taken from the mucosal side.\n\nRelatively well-demarcated yellowish mass without encapsulation is shown.\n\n(A) The tumor cells are composed of spindle cells with low nuclear atypia. (B) Nuclear palisading growth pattern is shown. (C) Lymphoid cuffing surrounding tumor cells is shown.\n\n(A) S-100 is diffuse, strong positive in tumor cells. (B) C-KIT is negative in tumor cells. (C) CD34 is negative in tumor cell, but normal vessel structures were stained. (D) SMA is negative in tumor cells, but normal smooth muscle in the proper muscle layer is stained.\n\nThe patient recovered from surgery uneventfully and was discharged on postoperative day 5. When she visited the out-patient clinic two weeks after discharge, she did not present any complication. No postoperative adjuvant therapy was performed.\n\n\nDiscussion\n\nSchwannomas are peripheral nerve sheath tumors which rarely develop in GI tract1,2. GI tract schwannomas represent about 2–6% of all mesenchymal tumors2,3.\n\nFor the first time, Daimaru et al. clarified the entity of the nerve sheath tumors developing in the GI tract and proposed these tumors to be designated as “benign schwannoma of the GI tract” in 19887. Lymphoid cuffing, benign nuclear atypia and positive immunostaining for S-100 protein were the distinct features of the schwannoma of the GI tract, which distinguish the schwannoma from other spindle-cell stromal tumors of smooth muscle origin7. Until the early 1990s, most GISTs traditionally had been classified as smooth muscle tumors8. Ueyama et al. suggested that most of the GIST had smooth muscle differentiation and excluded schwannomas from the GIST8.\n\nCurrently, GI tract schwannomas are classified as non-epithelial tumors of which disease entity is clearly distinct from leiomyomas, leiomyosarcomas, gastrointestinal autonomic nerve tumors (GANTs) and GISTs2,9. And GI schwannomas are considered distinguished from conventional soft-tissue schwannomas and CNS schwannomas2.\n\nGI schwannomas are mostly identified in the stomach and less frequently seen in colon, rectum, small intestine or esophagus2,3. They are most frequently diagnosed among people in their sixties and the incidence rates are identical for males and females3,9,10. Most of them are incidentally identified during screening endoscopy or imaging studies because they are usually asymptomatic. However, just like any other GI tumors, they can present some clinical symptoms such as abdominal pain, tenesmus, rectal bleeding or melena1. Sometimes these tumors manifest as colonic obstruction or intussusception4,9,10.\n\nEndoscopically, these tumors usually present as a submucosal tumor with smooth mucosa or with mucosal ulceration1–3. On CT scans, the tumors usually present as exophytic masses with homogeneous enhancement and cystic change, necrosis, or calcification within tumors are uncommon2.\n\nA preoperative diagnosis is challenging because endoscopic mucosal biopsy usually provides limited information to differentiate them from other mesenchymal tumors of GI tract such as GISTs, NETs, leiomyomas, or leiomyosarcomas3. In Inagawa’s study, only 15% of the colon schwannomas were diagnosed on preoperative endoscopic biopsy11; in Bohlok’s study, 24% of the colorectal schwannomas were diagnosed preoperatively3.\n\nDiagnosis is confirmed pathologically with immunohistochemical analysis. Histopathological features of schwannomas are mainly elongated bipolar spindle cells with variable cellularity and sometimes peripheral cuff-like lymphocyte infiltration is exhibited around the tumor, which helps to differentiate schwannomas from other spindle-cell tumors like fibromas or leiomyomas5,7,11. Schwannomas can be distinguished from other smooth-muscle tumors by strong s-100 positivity in immunohistochemical analysis11–13. Additionally, CD34 or c-kit protein is useful to distinguish the schwannomas from GISTs11–13. Schwannomas are S-100 positive, but CD-34 and c-kit negative; most GISTs are s-100 negative, but CD-34 and c-kit positive.\n\nPrognosis is generally promising because most of the GI schwannomas are benign and malignant potential is low1–3. However, even though many researchers reported the benign features of GI schwannoma, some of these tumors present local recurrence or distant metastasis. In Bohlok’s study, 3 (3.1%) out of 93 cases of colorectal schwannomas were malignant3. High mitosis rate, high Ki-67 index, and large tumor size are considered to be associated with malignancy3.\n\nComplete surgical resection obtaining free resection margins is the best therapeutic option1,3, because tumor recurrence is generally owing to incomplete surgical resection with inadequate margins2. In some limited cases, patients can be treated by endoscopic resection or transanal resection without undergoing radical surgery3.\n\nAdjuvant therapies are not commonly recommended if surgical resection achieving free margins is completed5,14. Currently, limitation of our knowledge is that there is no consensus for subsequent treatment after surgical resection in case of malignant transformation5.\n\nSigmoid colon schwannomas are very rare colonic neoplasms. To our knowledge, only 28 cases of sigmoid colon schwannomas have been published. Because of its rarity and challenge to diagnosis, review of the clinical features of the disease with a presenting case would be of help for physicians and surgeons. We believe that our study presents the clinical manifestations including endoscopic, imaging, histopathologic and immunohistologic findings of this rare disease with a thorough literature review and it provides guidance in diagnosis and treatment of the disease. Limitation of this study is that the treatment strategy for metastatic diseases was not suggested because only very limited cases were reported and no consensus exists for now.\n\n\nConclusion\n\nSigmoid colon schwannomas are usually found incidentally during screening colonoscopy and present as submucosal tumors. Preoperative diagnosis is challenging because clinical manifestations, as well as colonoscopic and CT findings, are nonspecific. No specific tumor marker exists either. Histopathologically, the tumors consist of spindle cells. However, colonoscopic biopsies have limitations in terms of a definite diagnosis and differential diagnosis includes schwannoma, GIST, NET, leiomyoma, leiomyosarcoma, etc. Conclusive diagnosis can be made by confirming s-100 proteins in immunohistochemical analysis and mostly confirmed post-surgically. Complete surgical resections with adequate free margins are required because although the majority of the diseases are benign, some are reported to be malignant. There is no consensus for adjuvant chemotherapy.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBaig MMAS, Patel R, Kazem MA, et al.: Schwannoma in the ascending colon, a rare finding on surveillance colonoscopy. J Surg Case Rep. 2019; 2019(2): rjz046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMekras A, Krenn V, Perrakis A, et al.: Gastrointestinal schwannomas: a rare but important differential diagnosis of mesenchymal tumors of gastrointestinal tract. BMC Surg. 2018; 18(1): 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBohlok A, El Khoury M, Bormans A, et al.: Schwannoma of the colon and rectum: a systematic literature review. World J Surg Oncol. 2018; 16(1): 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzález Ruiz Y, Reyes Delgado A, Guiterrez Alonso C, et al.: [Sigmoid intussusception as a clinical presentation of colonic schwannoma: Pediatric case]. Arch Argent Pediatr. 2019; 117(1): e68–e71. PubMed Abstract \| Publisher Full Text\n\nUhr A, Singh AP, Munoz J, et al.: Colonic Schwannoma: A Case Study and Literature Review of a Rare Entity and Diagnostic Dilemma. Am Surg. 2016; 82(12): 1183–86. PubMed Abstract\n\nÇakır T, Aslaner A, Yaz M, et al.: Schwannoma of the sigmoid colon. BMJ Case Rep. 2015; 2015: pii: bcr2014208934. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaimaru Y, Kido H, Hashimoto H, et al.: Benign schwannoma of the gastrointestinal tract: a clinicopathologic and immunohistochemical study. Hum Pathol. 1988; 19(3): 257–64. PubMed Abstract \| Publisher Full Text\n\nUeyama T, Guo KJ, Hashimoto H, et al.: A clinicopathologic and immunohistochemical study of gastrointestinal stromal tumors. Cancer. 1992; 69(4): 947–55. PubMed Abstract \| Publisher Full Text\n\nWilde BK, Senger JL, Kanthan R: Gastrointestinal schwannoma: an unusual colonic lesion mimicking adenocarcinoma. Can J Gastroenterol. 2010; 24(4): 233–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang WB, Chen WB, Lin JJ, et al.: Schwannoma of the colon: A case report and review of the literature. Oncol Lett. 2016; 11(4): 2580–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInagawa S, Hori M, Shimazaki J, et al.: Solitary schwannoma of the colon: report of two cases. Surg Today. 2001; 31(9): 833–38. PubMed Abstract \| Publisher Full Text\n\nMiettinen M, Virolainen M, Maarit-Sarlomo-Rikala: Gastrointestinal stromal tumors--value of CD34 antigen in their identification and separation from true leiomyomas and schwannomas. Am J Surg Pathol. 1995; 19(2): 207–16. PubMed Abstract \| Publisher Full Text\n\nRamai D, Lai J, Changela K, et al.: Transverse colon schwannoma treated by endoscopic mucosal resection: A case report. Mol Clin Oncol. 2017; 7(5): 830–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuraihi H, Assam JH, Sorrell M: Ascending Colon Schwannoma an Unusual Cause of Acute Lower Gastrointestinal Bleeding. S D Med. 2017; 70(1): 33–37. PubMed Abstract" }	[ { "id": "48558", "date": "31 May 2019", "name": "Huaibin Mabel Ko", "expertise": [ "Reviewer Expertise Gastrointestinal 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\nOverall this is a good case report and review of a rare tumor. There are a few small grammatical errors, but the paper is otherwise well organized and easy to follow.\nMinor suggestions:\nWhen referring to the size of the tumor, especially when giving dimensions that seem fairly exact (\"4.5 x 4.0 cm\") it is unnecessary to say \"about\", you can just state the size. Replace \"Most of the\" with \"Most\", as in \"Most GI tract schwannomas\" or \"Most colon schwannomas\" Figure 6: \"proper muscle layer\" should be \"muscularis propria layer\"\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": "54272", "date": "02 Oct 2019", "name": "Umut Riza Gunduz", "expertise": [ "Reviewer Expertise Breast cancer", "thyroid cancer", "endoscopy", "ERCP" ], "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 presented a case report and a review of related literature about sigmoid colon schwannomas. This is a rare disease and an interesting subject.\nAlthough it is a beautifully written review in general, some corrections are required:\nThe authors claimed segmenter resection is enough for sigmoid colon schwannoma but in their case report they prefer to anterior resection. it is necessary to explain this in the discussion section.\n\nThe authors shared a photo of the specimen they resected in figure 3. This image resembles segmental resection rather than anterior resection.\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/8-652
https://f1000research.com/articles/8-651/v1	13 May 19	{ "type": "Research Article", "title": "Structural, functional and docking analysis against Schistosoma mansoni dihydroorotate dehydrogenase for potential chemotherapeutic drugs", "authors": [ "Benson Otarigho" ], "abstract": "Background: Praziquantel, as the only drug for the treatment of schistosomiasis, is under serious threat due to the emergence of resistant strains of Schistosoma species. There is an urgent need to search for alternative chemotherapy to supplement or complement praziquantel. Schistosoma dihydroorotate dehydrogenase (DHODH) has been recommended as a druggable target for schistosomiasis chemotherapy. The development of novel molecular modeling approaches, alongside with computational tools and rapid sequencing of pathogen genomes, have facilitated drug discovery. Therefore, the aim of this study was to employ computational approaches to screen compounds against Schistosoma mansoni DHODH. Methods: In this study, DHODH was used to blast on the latest version of DrugBank that contained 12,110 compounds, resulting in 26 drugs that can bind. Results: In silico docking shows that 13 drugs can bind strongly with an estimated free energy of binding, total intermolecular energy and estimated inhibition constant (Ki) greater than or equal to -8.6 kcal/mol, -8.12 kcal/mol and 1.12 µM, respectively. These compounds include the approved drugs manitimus, capecitabine, brequinar analog and leflunomide. Conclusions: These results indicate that these drugs have the potential for use in the control of schistosomiasis in the future.", "keywords": [ "Dihydroorotate Dehydrogenase", "Schistosomiasis", "Drugs", "Neglected Tropical Disease" ], "content": "Abbreviations\n\nDHODH: dihydroorotate dehydrogenase, SmDHODH: Schistosoma mansoni dihydroorotate dehydrogenase, SchDHODH: Schistosoma dihydroorotate dehydrogenase, ShDHODH: Schistosoma haematobium dihydroorotate dehydrogenase, SjDHODH: Schistosoma japonicum dihydroorotate dehydrogenase, HsDHODH: Homo sapien dihydroorotate dehydrogenase, MmDHODH: Mus musculus dihydroorotate dehydrogenase, RrDHODH: Rattus rattus dihydroorotate dehydrogenase, NTD: neglected tropical disease, PLIs: protein-ligand interactions\n\n\nIntroduction\n\nThe public health impacts of schistosomiasis, caused by Schistosoma species, are second only to malaria in endemic regions and it is an important neglected tropical disease (NTD)1,2. More than 240 million people are infected with one or more Schistosoma species in the tropical or subtropical regions2–4. Approximately 85% of these infections occur in sub-Sahara Africa3,4. Movement of refugees and mass migration of individuals from endemic to non-endemic areas have recently expanded the areas at risk of schistosomiasis and increased disease prevalence in recent times5–7. Around 200,000 people die from Schistosoma infections each year and many more suffer with serious disabilities. Moreover, there is great economic loss in areas where schistosomiasis is endemic. Reports have shown that over 120 million people are symptomatic, with 20 million having severe clinical disease, in endemic areas1,2,4. The disease has been reported to be endemic in 76 countries, in which approximately 4 billion people live. Involvement in agricultural work, domestic chores and recreational activities can expose them to infested water1,2,8.\n\nThe control of this disease is solely dependent on the chemotherapeutic drug praziquantel, which is used to treat infected individuals. There is no effective vaccine currently3,9. Other chemotherapy alternatives include oxamniquine and metrifonate, although these have some setbacks, such as being ineffective against all life stages of the parasite and severe side effects, which is why praziquantel is recommended10–17. Although praziquantel is effective, relatively safe and low cost, laboratory and field studies have shown the emergence of a resistant parasite strain in certain regions. This has led to a serious search for chemotherapeutic alternatives for the treatment and control of schistosomiasis9,10. Therefore, there is a need to search for alternatives using both laboratory and computational investigative methods to explore sequenced Schistosoma genomes9.\n\nThe recent sequencing of Schistosoma genomes, alongside the quickly evolving field of bioinformatics, has unveiled numerous opportunities to identify and characterize important proteins, which can aid in the development of novel drugs for disease control. The application of bioinformatics tools in extracting significant meaning from the sequence data have improved our knowledge of the mode of action of proteins18. Modeling protein-ligand interactions (PLIs) to determine the possible biological interactions that occur in vivo has helped in understanding protein function and predicting their mechanisms of action. PLIs are also important in the development of novel therapies and diagnostic tools19–21.\n\nDihydroorotate dehydrogenase (DHODH) is a crucial enzyme that catalyzes the conversion of dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway22–25. Schistosoma DHODH has been highly recommended as a druggable target that could offer alternative routes for schistosomiasis control22,24. Detailed structural differences have been demonstrated between human and Schistosoma DHODH9. Furthermore, Schistosoma mansoni cannot synthesize purine bases de novo, hence depends exclusively on the salvage pathway.\n\nIn this study, the Schistosoma DHODH sequences were thoroughly explored and compounds from an available drug database were docked against this enzyme to assess their suitability as potential drugs. The results show that 13 approved drugs used in the treatment of other diseases have the potential to inhibit the activities of SmDHODH.\n\n\nMethods\n\nSchDHODH protein sequences were obtained from the NCBI protein database. The sequences were confirmed in SchistoDB for S. haematobium (two protein sequences) and GeneDB for S. mansoni (two protein sequences) and S. japonicum (one protein sequence). All sequences were obtained in FASTA format. Each of these protein sequences were used for a BLASTp search of the non-redundant protein database on NCBI, employing the protein-protein BLAST algorithm. Results with similarities above 50% and with a query coverage and expectation value of >70% and 0.0, respectively, were obtained and combined for phylogenetic tree construction. Hypothetical and unknown proteins were excluded, even when there was high identity. After combining these sequences, duplicate protein sequences were also excluded. Host DHODH protein sequences were also obtained from NCBI using the search terms as targets “Homo sapiens + DHODH”, “Mus musculus + DHODH” and “Rattus rattus + DHODH” separately. The protein sequences that had 98% similarity to the targets were retrieved for further analyses. One HsDHODH, one MmDHODH and two RrDHODH sequences were retrieved. Each SchDHODH sequence was used to search DrugBank (version 5.1.2, released 2018-12-20)26 for potential compounds that may bind. All settings were at default.\n\nFunctional domains for each SchDHODH, MnDHODH and HsDHODH were computed using the normal and genomic mode of SMART27,28. The functional domains predicted were validated using other webtools; NCBI Conserved Domains29–32, PROSITE33, InterPro34 and Pfam version 32.035. Default parameters were used for these analyses.\n\nSince, for many disordered proteins, binding affinity with their receptors is regulated by post-translational modification, SmDHODH protein was analyzed for intrinsically disorder. SmDHODH was the only protein used in this analysis because it was used for modeling and protein-drug interaction. PONDR, which can predict natural disordered regions in a protein sequence, was used. This was validated using other similar tools such as SLIDER webserver36 and DisEMBL Intrinsic Protein Disorder Prediction 1.537.\n\nEach protein’s physiochemical properties, which includes the number of residues, molecular weight and extinction coefficient were predicted using the ProtParam web tool38. These properties were validated using the PepCalc peptide property calculator and Protein Physicochemical Properties Prediction Tool (PPPPT) web tools.\n\nAll SchDHODH, HsDHODH and MmDHODH sequences were compared by Multiple Sequence Alignment (MSA) and the conserved and deleted regions were analyzed. The MSA analysis was carried out using Clustal Omega tools in Jalview39. The MSA was analyzed for conserved properties and regions of similarity.\n\nPhylogenetic trees were constructed using MEGA version 7 software40. The constructed phylogenetic trees were validated using Phylogeny.fr41. The tree file, in Newick format, was exported and visualized in FigTree software version 1.4.242 for proper annotation. The pairwise distances were also estimated, using the Poisson correction model in MEGA40.\n\nProtein-protein interactions were determined using the STRING database for functional protein association networks. Each of the SchDHODH, HsDHODH and MmDHODH protein sequences were searched on the STRING and the interactions were downloaded in jpeg format.\n\nSmDHODH sequences were modelled using SWISS-MODEL43–47. The template 3u2o.1.A, with a sequence identity of 47.95%, GMQE of 0.74 and QMEAN of -0.98 was selected as the model of choice for SmDHODH sequences.\n\nMolecular docking was carried out using DockingServer48 and Gasteiger partial charges were added to the ligand atoms49, as described by Kumar, 201150. Proteins were uploaded, protein charges were calculated, and solvation parameters were calculated and cleaned. All advance docking parameters were left at default.\n\n\nResults\n\nIn total, five SchDHODH sequences were retrieved for further analyses. One for S. japonicum and two proteoforms each for S. haematobium and S. mansoni. One HsDHODH sequence, two MmDHODH sequences and two RrDHODH sequences (Figure 1 and Project 1, Extended data)51 were included in the study for proper comparison as mammals, including humans, are the hosts, while rats and mice are commonly used in the laboratory for schistosomiasis studies. Of 12,110 drugs in the database, 26 compounds that can bind to SchDHODH proteins were identified and retrieved from DrugBank. These drugs were retrieved with E value: 7.46761e-71, Bit score: 224.172, query length: 379 and alignment length: 314. These results were the same for each of the SchDHODH sequences searched. All of the drugs were at the experimental phase, except flavin mononucleotide, capecitabine and leflunomide, which are approved for the treatment of other diseases, and manitimus, which is still at the investigational phase. The mode of action of most of these retrieved drugs are not known, except for atovaquone, leflunomide and teriflunomide, which act as inhibitors to known proteins other than DHODH.\n\nConserved amino acids across SchDHODHs are marked and indicated in purple boxes, while across SmDODHs and ShDHODHs are marked and indicated in red boxes. The red arrows show where the mutation in SjDHODH compare to SmDODHs and ShDHODHs.\n\nThe protein domain analysis (see Project 3, Extended data)52 shows that all analyzed proteins have the dihydroorotate dehydrogenase domain (DHO_dh), while ShDHODH proteins have dynein light domains and transmembrane helix regions, which are not seen in the other proteins. The disorder predictions show that a small fraction of the SmDHODH protein is disordered, as shown in Project 2, Extended data53.\n\nPhysiochemical parameters (see Project 1, Extended data)51 and alignment (Figure 1), as well as phylogenetic analyses of all the SchDHODH sequences (Figure 2), show that the DHODH proteins of S. haematobium and S. mansoni could be evolutionary closer than that of S. japonicum. However, S. japonicum evolved 0.8928 million years ago (mya), compared to S. haematobium which evolved 0.8207 mya and S. mansoni which evolved 0.8012 mya. The last common ancestor for DHODH of both S. haematobium and S. mansoni was 0.8379 mya. The host DHODHs evolved more recently, as shown in Figure 2. These results could also explain the similarities between DHODHs that are shown in the sequence alignment (Figure 1). Mutations that occurred in ShDHODHs were also observed in the SmDHODHs, though there are some points where all three Schistosoma species shared mutation points. However, the similarities between ShDHODH and SmDHODH sequences do not correlate with the physiochemical parameters; ShDHODH theoretical pI, extinction coefficients, instability index, aliphatic index and grand average of hydropathicity scores are more similar to SjDHODH than SmDHODH (see Project 1, Extended data)51.\n\nAll the SchDHODHs shows clearly that the SchDHODHs of S. haematobium and S. mansoni could be evolutionary closer compared to S. japonicum.\n\nThe prediction of possible in vivo interactions of these DHODHs showed that SchDHODH binds to NADPH (gene Smp_166580), putative glutamate synthase (Smp_128380.2) and NADH-cytochrome B5 reductase (Smp_053230) (Figure 3). These four proteins bind to each other; however, it is unclear whether they form a complex. SchDHODHs have an inhibitory effect on orotate phosphoribosyltransferase (Smp_050540), which has an inhibitory transcriptional regulation on aspartate carbamoyltransferase (Smp_186670) and vice versa. HsDHODH and MmDHODHs non-specifically bind to collapsin response mediator protein 1 (Crmp1) and different proteoforms of dihydropyrimidinase (Dpys). However, HsDHODH and MmDHODHs have an inhibitory role on carbamoyl-phosphate synthetase (Cps1) and uridine monophosphate synthetase (Umps).\n\nInteraction of proteins with (a) SmDHODH (b) HsDHODH and (c) MmDHODH.\n\nSmDHODH was selected for modeling and protein-drug interaction studies as a representative for the three SchDHODHs, since sequence alignment and phylogenetic analysis showed that they are all closely related. The modelled SmDHODH used for docking against the 26 potential compounds is shown in Figure 4. After the molecular docking, 13 drugs had a binding affinity of less than -6kcal/mol, suggesting a strong bond21, with SchDHODH (Table 1 and Figure 5) and details of these interactions are shown in Figure 6. The results show that 3-{[(3-fluoro-3'-methoxybiphenyl-4-yl)amino]carbonyl}thiophene-2-carboxylic acid (accession number DB07976) and 3-{[(3-fluoro-3'-methoxybiphenyl-4-yl)amino]carbonyl}thiophene-2-carboxylic acid (accession number DB07978) have the highest binding affinity, with an estimated free energy of binding (kcal/mol) of -10.18 and -10.66, respectively. Leflunomide (accession number DB01097) and N-anthracen-2-yl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (accession number DB08006) both have an estimated free energy of binding of -8.12. The SchDHODH protein residues that interact with the different drugs in Table 1, are shown stick while drugs cartoon. It was observed that SmDHODH interacts with the various compounds using hydrogen bonds, polar, hydrophobic, pi-pi, halogen and other forms of bonding.\n\na) 2-({[2,3,5,6-TETRAFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENTA-1,3-DIENE-1-CARBOXYLIC ACID (Accession Number: DB07978); b) 3-{[(3-FLUORO-3'-METHOXYBIPHENYL-4-YL)AMINO]CARBONYL}THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07976); c) 3-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07977); d) Flavin mononucleotide (Accession Number: DB03247); e) 2-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENT-1-ENE-1-CARBOXYLIC ACID (Accession Number: DB07975); f) 5-Carbamoyl-1,1':4',1''-terphenyl-3-carboxylic acid (Accession Number: DB04583); g) 5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08008); h) Manitimus (Accession Number: DB06481); i) Capecitabine (Accession Number: DB01101); j) Brequinar Analog (Accession Number: DB03480); k) (2Z)-N-biphenyl-4-yl-2-cyano-3-cyclopropyl-3-hydroxyprop-2-enamide (Accession Number: DB08169); l) Leflunomide (Accession Number: DB01097); m) N-anthracen-2-yl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08006).\n\na) 2-({[2,3,5,6-TETRAFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENTA-1,3-DIENE-1-CARBOXYLIC ACID (Accession Number: DB07978); b) 3-{[(3-FLUORO-3'-METHOXYBIPHENYL-4-YL)AMINO]CARBONYL}THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07976); c) 3-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07977); d) Flavin mononucleotide (Accession Number: DB03247); e) 2-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENT-1-ENE-1-CARBOXYLIC ACID (Accession Number: DB07975); f) 5-Carbamoyl-1,1':4',1''-terphenyl-3-carboxylic acid (Accession Number: DB04583); g) 5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08008); h) Manitimus (Accession Number: DB06481); i) Capecitabine (Accession Number: DB01101); j) Brequinar Analog (Accession Number: DB03480); k) (2Z)-N-biphenyl-4-yl-2-cyano-3-cyclopropyl-3-hydroxyprop-2-enamide (Accession Number: DB08169); l) Leflunomide (Accession Number: DB01097); m) N-anthracen-2-yl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08006).\n\n\nDiscussion\n\nIn the present study, known drugs that are either approved for treatment or at the experimental stage were searched as targets of DHODHs. Of those searched, 13 had a strong binding affinity. Binding affinity of less than -6kcal/mol is regarded a strong bond21. The strength of hydrogen, polar, pi-pi, halogen and hydrophobic bonding between the drugs and the SmDHODH shows the stability of the ligand inside the binding pocket. Based on the interaction properties of the ligand-protein complex, all the drugs have strong affinity, however, drug DB07978 (2-({[2,3,5,6-tetrafluoro-3'-(trifluoromethoxy)biphenyl-4-yl]amino}carbonyl)cyclopenta-1,3-diene-1-carboxylic acid) and DB07976 (3-{[(3-fluoro-3'-methoxybiphenyl-4-yl)amino]carbonyl}thiophene-2-carboxylic acid) have the strongest free energy of binding among all the drugs screened.\n\nOne of the drugs with a high affinity (estimated free energy of binding of -9.56) to SmDHODH is flavin mononucleotide (accession number DB03247) (vitamin B2). This compound is naturally present in most food and used as a food additive54–57. Riboflavin supplementation has been known to increase the efficiency of cell energy metabolism58,59. Manitimus (accession number DB06481) was also identified by the docking experiment as a potential drug for schistosomiasis. It is an efficacious and well tolerated drug for kidney transplant patients. It is a novel compound with multiple mechanisms of action60,61. One mechanism is via the suppression of de novo pyrimidine biosynthesis, inhibiting the action of dihydroorotate dehydrogenase and consequently inhibiting cell proliferation62.\n\nAnother promising drug is capecitabine (accession number DB01101), which is a chemotherapy medication used to treat numerous types of neoplasms, including those of the breast, esophagus, larynx and gastrointestinal and genitourinary tracts. Capecitabine is a prodrug and is enzymatically converted to fluorouracil (an antimetabolite) in the tumor, where it inhibits DNA synthesis and slows growth of tumor tissue63–65. Leflunomide (accession number DB01097) is an approved immunosuppressive disease-modifying antirheumatic drug that has been in use for the treatment of rheumatoid arthritis and psoriatic arthritis for more than 10 years. Its mode of action is via pyrimidine synthesis by inhibiting dihydroorotate dehydrogenase26,66–70.\n\n\nConclusions\n\nThere is an urgent need to search for and develop novel drugs that can be used in the treatment of schistosomiasis to complement treatment with praziquantel as there are growing number of reports of praziquantel-resistant Schistosoma strains in the laboratory as well as in the field. Furthermore, there are no efficient vector control strategies. SmDHODH has been proposed as druggable target in the de novo pyrimidine biosynthesis pathway for schistosomiasis chemotherapy. We used the SmDHODH sequence to search for all possible inhibiting compounds from the DrugBank database and found 13 with the potential to bind efficiently. If these compounds, which are already approved or in experimental stages, are tested in a wet laboratory experiment against the Schistosoma parasite and any are found to be effective, they could be easier than novel compounds to approve to supplement praziquantel for the control of schistosomiasis in the future.\n\n\nData availability\n\nSJCHGC02326 protein [Schistosoma japonicum], Accession number AAW26221: https://identifiers.org/ncbiprotein/AAW26221\n\nDihydroorotate dehydrogenase (quinone), mitochondrial [Schistosoma haematobium], Accession number KGB36135: http://identifiers.org/ncbiprotein/KGB36135\n\nDihydroorotate dehydrogenase (quinone), mitochondrial [Schistosoma haematobium], Accession number XP_012795900: https://identifiers.org/ncbiprotein/XP_012795900\n\ndihydroorotate dehydrogenase [Schistosoma mansoni], Accession number CCD78646: http://identifiers.org/ncbiprotein/CCD78646\n\ndihydroorotate dehydrogenase [Schistosoma mansoni], Accession number XP_018651255: https://identifiers.org/ncbiprotein/XP_018651255\n\ndihydroorotate dehydrogenase (quinone), mitochondrial precursor [Mus musculus], Accession number NP_064430 : http://identifiers.org/ncbiprotein/NP_064430\n\nChain A, DIHYDROOROTATE DEHYDROGENASE [Rattus rattus], Accession number 1UUM_A: http://identifiers.org/ncbiprotein/47169292\n\nChain B, DIHYDROOROTATE DEHYDROGENASE [Rattus rattus], Accession number 1UUM_B: http://identifiers.org/ncbiprotein/47169293\n\nCrystal structure of human dihydroorotate dehydrogenase at 1.7 A resolution [Homo sapiens], Accession number 5K9D: https://identifiers.org/pdb/5K9D\n\nFigshare: Extended data 1_Physiochemical Properties.docx. https://doi.org/10.6084/m9.figshare.8019683.v151\n\nFigshare: Prediction of intrinsic disorder of SmDHODH.docx https://doi.org/10.6084/m9.figshare.8050541.v153\n\nFigshare: Extended data 3_Protein domain analysis of DHODHs of the Schistosoma sp and Host.docx https://doi.org/10.6084/m9.figshare.8051777.v152\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMakamu F, Azam M, Kazianga H: Returns to Controlling a Neglected Tropical Disease: Schistosomiasis Control Programme and Education Outcomes in Nigeria. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "50744", "date": "15 Jul 2019", "name": "Madhu Saddala", "expertise": [ "Reviewer Expertise Bioinformatics", "Genomics", "Proteomics", "Microarray", "Single cell sequencing", "miRNA", "noncoding RNAs", "drug 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 article provided by Benson provides a clear conceptual description of the structural, functional and docking analysis and potential Schistosoma mansoni dihydroorotate dehydrogenase chemotherapeutic drugs. The work follows cheminformatic standards for drug development, and demonstrates the physical characteristics of the SchDHODHs, HsDHODHs and MmDHODHs identified as potential small molecule inhibitors of Schistosoma mansoni dihydroorotate dehydrogenase. The rationale, testing, and work supporting the compounds is well described and clear to follow, and the rationale for searching for Schistosoma mansoni dihydroorotate dehydrogenase inhibitors is well explained. The scope of the article is narrow, in that it focuses on lead cheminformatic validation, but it is relevant to several potential audiences and is rationally screened through those pipelines. Additional in vitro validation would raise the impact of these compounds, but may not be necessary to report them as a suitable candidate for follow-on work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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? Yes", "responses": [] }, { "id": "50743", "date": "22 Jul 2019", "name": "Elumalai Pavadai", "expertise": [ "Reviewer Expertise Computer-Aided Drug Discovery", "Chemoinformatics", "Bioinformatics", "Molecular 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\nIn the study, Otarigho has reported the structural and physical characterization of Schistosoma DHODH enzyme with the use of different computational prediction methods including, protein-protein interaction and homology modeling. In addition, the author has identified hit compounds by structure-based virtual screening of DrugBank database and suggested some compounds as possible SmDHODH inhibitors. Overall, this manuscript is well-written, and the analyses appear well done. However, it looks incomplete without the experimental validations of the compounds and also lacks some results/discussion as commented below, and the author should address them in a revised manuscript.\nNo crystal structures are available for any Schistosoma DHODHs and some differences have been shown in the active of DHODH as given in Fig 1. I suggest the author build models for other Schistosoma DHODHs mentioned in the manuscript, in addition to the SmDHODH, and compare the active sites between human and Schistosoma DHODHs. This can help us to understand the selectivity of Schistosoma DHODH inhibitors. In addition, there are some Schistosoma DHODH models have been published and the author may also want to compare the models with the published ones.\n\nTo make sure the employed docking protocol is valid and reliable, the author may want to perform docking of some Schistosoma DHODH inhibitors reported previously before performing the docking of the DrugBank compounds. The author can check whether the docking protocol correlates the biological activity of the compounds or not.\n\nSome malarial and human DHODH inhibitors are in clinical trials. The author needs to make sure whether the screening resulted in any DHODH compounds. This needs to be discussed in detail in the manuscript.\n\nRegarding comment 3, I can see some human and malarial DHODH inhibitors in Fig 5. Can the author dock them into respective crystal structures and compare with Schistosoma DHODHs? I believe some of the compounds have been crystallized.\n\nThere are some similar compounds in Fig 5. For example, g and m are sharing the same scaffold, triazolopyrimidine. I suggest the author remove the similar compounds and consider structurally diverse ones.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "50745", "date": "25 Jul 2019", "name": "Maria Cristina Nonato", "expertise": [ "Reviewer Expertise structural biology", "medicinal chemistry", "enzymology" ], "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 manuscript prepared by Dr Benson Otarigho describes an in silico study to characterize the enzyme dihydroorotate dehydrogenase (DHODH) from different Schistosoma species, and compare them with their orthologues from humans, mice and rats. Moreover, docking studies have been performed by evaluating the DrugBank library. Based on the findings described, out of the 12110 compounds tested, 26 compounds have been identified as Schistosoma DHODH binders, where 13 of them are approved drugs.\n\nOverall, I am not convinced that the work presents original findings. Moreover, there are many aspects in terms of methodology and interpretation of the results that should be carefully revised. My comments are based on the following points:\nDHODH is a very well characterized enzyme, including Schistosoma DHODH. Functional domain analyses and physical and chemical properties studies described in the manuscript, all data raised by online/automatic tools, do not bring any new contribution to the field.\n\nMoreover, several studies reported were based on amino acid sequences retrieved from different sources, including, for example, sequences retrieved from the protein data base. In this case, for example, the sequence for human and rat DHODHs (Figure 1) are found truncated. This, of course, was necessary for the crystallographic studies, but it can not be used for phylogenetic studies, analysis of intrinsic disordered regions, and analysis of protein-protein interaction. In fact, the use of the truncate sequences took the author to draw wrong conclusions such as: \"The protein domain analysis (see Project 3, Extended data) shows that all analyzed proteins have the dihydroorotate dehy-drogenase domain (DHO_dh), while ShDHODH proteins have dynein light domains and transmembrane helix regions, which are not seen in the other proteins\" First, the class 2 DHODH, including human and Schisto enzymes, possesses two domains: the dihydroorotate dehydrogenase domain and the membrane binding domain. It is important to describe both domains in order to differentiate class 1A and class 2 DHODH enzymes. Moreover, and very important, human/rat enzymes also possess the transmembrane helix regions, which was found omitted in the sequence retrieved from PDB. Figure 1 is overall very confusing. First the legend does not match with its content. Moreover, from Figure 1 one can not know which sequence refers to each organism.\n\nRegarding the 3D model:\nThe author used the online tool SWISS-MODEL to automatically generate a 3D model for Schistosoma DHODH. Since this model was used as a template for the docking experiments, more information regarding the quality of the model should be provided. Moreover, other features of the model that directly impact on the docking results should be carefully presented and discussed. For instance: Schistosoma DHODH has an insertion of approximately 10 residues compared to all the previous described class 2 DHODHs.\n\nHow has SWISS-MODEL dealt with that? Has the model included the FMN?\n\nRegarding molecular docking:\nWhich proteins have been uploaded for docking?\n\nWhere were the compounds found to bind Schistosoma DHODH? Are they found to bind in the \"quinone binding site\"? Even FMN?\n\nFigure 6 does not provide any information regarding the interaction between DHODH and the ligands. A picture prepared by LIGPLOT, for instance, can be more appropriated to describe the environment of a ligand bound to the protein.\n\nLeflunomide is a prodrug found to bind Schistosoma DHODH. Lefluomide is well known to display little or no effect on class 2 DHODH. An experimental evidence of this unexpected finding should be provided. Several other molecules have been identified. Some of them are already described as class2 DHODH inhibitors, others have not. For those, experimental evidences of such binding should be provided.\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?\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-651
https://f1000research.com/articles/8-80/v1	21 Jan 19	{ "type": "Research Article", "title": "Tobacco smoking and nicotine delivery alternatives: patterns of product use and perceptions in 13 countries", "authors": [ "Farhad Riahi", "Sarah Rajkumar", "Derek Yach", "Farhad Riahi", "Derek Yach" ], "abstract": "Background: Smoking tobacco products remains a significant public health problem. The Foundation for a Smoke-Free World commissioned a 13-country survey to gain a clearer understanding of the current landscape of smoking behavior and preferences across the world. Methods: Over 17,000 participants in 13 countries, representing different regions and income groups, answered questions on their smoking patterns and product use, their social context, their motivation to smoke, quit, or switch, and their perception of risks of products and substances. Rim weighting was done for each country to align responses with population demographics, and an additional 200 smokers for each country were surveyed to achieve sufficient sample size for sub-analyses of smoker data. Results: The observed prevalence of smoking ranged from an age-adjusted high of 57.5% in Lebanon to 8.4% in New Zealand among men, with lower rates for women. The majority of smokers were between 25-54 years old, had daily routines and social patterns associated with smoking, used boxed cigarettes, and rated their health more poorly compared to never smokers. Among a range of products and substances, smokers tended to give both cigarettes and nicotine the highest harm ratings. Smokers in high income countries were largely familiar with electronic nicotine delivery systems; the most commonly given reasons for using them were to cut down or quit smoking. A majority of smokers had tried to quit at least once, and while many tried without assistance, motivations, intentions, and methods for smoking cessation, including professional help, nicotine replacement therapies or medications, or electronic cigarettes, varied among countries. Conclusions: Smoking is deeply integrated in smokers’ lives worldwide. Although a majority of smokers have tried to quit, and are concerned for their health, they do not seek help. Smokers lack understanding of the harmful components of smoking tobacco products and the risk profile of alternatives.", "keywords": [ "tobacco", "smoking", "cigarettes", "quitting", "nicotine replacement therapy", "electronic nicotine delivery systems" ], "content": "Introduction\n\nWhile the worldwide rate of tobacco smoking has declined substantially in recent years, the absolute number of people currently smoking has increased from approximately 720 million smokers in 1980 to an estimated 1.1 billion today, the consequence of population growth outpacing declining smoking prevalence in many low and middle income countries1–3. Additionally, there are significant differences between countries in terms of the epidemiology of smoking and tobacco product preferences.\n\nThe ramifications of smoking are well-known. In 2015, smoking was the second leading risk factor for death and disability worldwide and accounted for 11.5% of the world’s deaths and 6.0% of global disability-adjusted life years4. Of the 7.1 million deaths attributed to tobacco use in 2016, 6.3 million were from cigarette smoking5.\n\nThe majority of smokers say they want to quit. An analysis of 10 years of National Health Interview Surveys in the United States (US) reported that in 2015, 68% of smokers wanted to quit and 55.4% had tried to quit within the previous year; however, only 7.4% were successful in quitting that year despite a range of available smoking cessation counseling and pharmacologic options6.\n\nIn recent years, the use of electronic nicotine delivery systems (ENDS) such as e-cigarettes have gained popularity in many high-income countries, with some evidence that they are being used as tools to reduce or quit smoking7. Although the health effects of these products are still under investigation, some public health experts suggest they may be used as harm reduction products and smoking cessation tools8–10.\n\nTo tackle the enormous global health smoking crisis effectively, more information is needed on the behavior and perceptions of smokers, ex-smokers, and never-smokers towards tobacco products and alternatives. The Foundation for a Smoke-Free World (FSFW) commissioned one of the largest global surveys of smoking habits in order to glean a better understanding of the current landscape of tobacco product use, the population’s grasp of the harm caused by different tobacco products and alternatives, reasons for smoking and for trying to quit or switch, as well as choices of smoking cessation methods.\n\n\nMethods\n\nThe FSFW commissioned the consulting and research agency Kantar Public to develop and execute a global survey on adult smoking in 13 countries: Brazil, France, Greece, India, Israel, Lebanon, Japan, Malawi, New Zealand, Russia, South Africa, the United Kingdom (UK), and the US. These countries were selected to represent a variety of markets in terms of income level, smoking prevalence, and smoking habits.\n\nKantar Public developed an 81-question quantitative survey based on existing publications and publicly available data on smoking habits and perceptions. The survey covered four domains: epidemiology of smoking and product use; social context of smokers; motivation to smoke, quit, or switch; and risk perception of products and substances. The survey and sources used are available. Kantar Public upholds the best market research industry practices and all personal data were anonymized so personal data no longer related to identifiable persons and subjects cannot be reidentified. All respondents were volunteers and gave oral (personal interview) or written (online surveys) consent. Oral consent was obtained as some participants may have limited proficiency in reading and writing. Before answering the survey, all respondents received information on what the research was about, what their participation in the project entailed, and any risks involved. Due to the low-risk nature of the study design, the FSFW and Kantar Public did not seek Institutional Review Board approval for the survey.\n\nThe survey was piloted with telephone interviews targeting two smokers, two ex-smokers, and two never smokers in each of the 13 countries. Pilot survey respondents were nonrandomly chosen from a contact list based on their smoking status. Smokers were defined as those who responded that they currently smoked cigarettes, cigars, cigarillos or a pipe “regularly” or “occasionally;” ex-smokers were defined as those who responded that they used to smoke but stopped; and never smokers were defined as those who responded that they had never smoked.\n\nThe sampling plan of the main sample was designed to be nationally representative of all adult citizens (18+) living in the country. Persons below 18 years were excluded from the survey.\n\nIn France, Israel, Japan, New Zealand, the UK, and the US, where potential responders in each stratum could be reached via a generic email invitation, respondents completed the survey online in their native language. Participants were stratified according to the most up-to-date census data, with quota definitions based on gender, age, and region to ensure that survey results represented the most accurate estimations of the target populations. Online panels depend on non-probabilistic sampling procedures, in which potential respondents voluntarily sign up to participate in the panel in general and in the survey in particular, which might induce a certain self-selection bias. In order to limit such bias, a large and diverse sampling frame and an effective sampling procedure were set up, sending out only generic survey invitations that did not give any indication of the topic.\n\nParticipants in seven countries (Brazil, Greece, India, Lebanon, Malawi, Lebanon, Russia) where email outreach would be inadequate answered the survey face-to-face. The interviewers used validated scripts in the participant’s native language. A stratified random probability sampling approach was used for the interviews. A unit selection performed at each step of the sampling process ensured a completely random approach. Based on the official population statistics, a certain number of primary sampling units (PSUs) were selected randomly, covering both urban and rural areas. According to the overall target sample, the number of interviews per PSU was calculated. In urban areas, a specific street was chosen randomly; in rural areas, the sampling point was selected randomly either from a list of streets (if such a list was available) or from a list of landmarks (church, library, bus stop, etc.). Households were selected using a random route procedure. In urban areas and in rural areas where a list of streets was available, the household with the lowest number in the street was selected as the sampling point. In the other rural areas, the household closest to the chosen landmark was selected as the starting point. After a successful interview, five households were skipped in urban areas and three in rural areas. After unsuccessful interview attempts, the interviewer simply proceeded to the next household without skipping. Within a household, individual respondents were selected using the recent birthday method (the interview was carried out with the adult in the household who had the most recent birthday). Three attempts were made to complete the interview with the selected respondent before proceeding to the next household. Quotas were set as independent response targets for each characteristic: targets were pursued per class within each variable, regardless of achievement of the other quota variables.\n\nThe survey was conducted between October 27, 2017 and December 30, 2017. The number of completed interviews was between 700 and 3,200 respondents per country, proportional to the population size. Kantar Public oversampled 200 additional smokers in each country to allow a more detailed analysis of the results for smokers. A total of 17,160 smoking and non-smoking participants completed the survey, 10,298 in face-to-face interviews and 6,862 online.\n\nData analyses were done using SPSS (IBM Corp, Version 24). Descriptive analyses were calculated for all variables. A rim weighting procedure was run against the population figures to construct weight variables, with the procedure executed separately for each country. Rim weighting consists of iterations: sample counts for each weight variable were adjusted to fit the actual population proportions (marginal percentages) using as the initial values the result of the previous adjustment. The weighting strategy was designed to correct any misbalance following field work in terms of the three original quota targets (age, gender, and region). The statistical z-test was used to find significant differences in proportions among independent samples of smokers and never-smokers.\n\n\nResults\n\nThe observed prevalence of smoking in adults 18 years and older in this survey ranged from an age-adjusted high of 57.5% in Lebanon to 8.4% in New Zealand among men, and from 48.4% in Lebanon to 1.0% in India among women (Table 1). The majority of smokers in all countries were between the ages of 25 and 54 (Table 2).\n\nData are presented as unweighted n and weighted percentages\n\nBR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States\n\nData are presented as unweighted n and weighted percentages\n\nBR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States\n\nBoxed cigarettes were the preferred tobacco product of choice in all countries, followed by hand-rolled cigarettes/roll-your-own (RYO) in most countries (Figure 1). In almost all countries, the majority of smokers surveyed (47-91%) viewed themselves as light or moderate smokers (Figure 2). Between 58% (Israel) and 94% (Greece) smoked daily (Figure 3).\n\nSmokers: Which of the following tobacco products do you use? (multiple answers possible). % top 3 answers. Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nData are presented as smokers unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States. Column percentages may not add up to 100% due to rounding.\n\nData are presented as smokers unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States. Column percentages may not add up to 100% due to rounding.\n\nIn aggregated data across all countries, a majority of smokers smoked after meals (62.2%), and many also smoked every time they had coffee or tea (46.1%), or an alcoholic beverage (43.6%). Smokers were also tempted to smoke when they saw others smoking nearby (41.9%). Figure 4 shows the breakdown for these routines by country.\n\nIn almost all countries, more current smokers than ex-smokers or nonsmokers were surrounded by people who also smoked, including parents, spouse/partner, close friends and colleagues (Figure 5a-d).\n\nSmokers, multiple answers possible, % yes. Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nData are presented as unweighted n and weighted percentages for % applicable. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nNearly two-thirds or more of smokers in all countries consider themselves addicted to cigarettes (Table 3). Smokers were also asked if they smoked a few minutes after waking up (15% Israel to 59% Malawi) and if they could not go two hours without smoking (13% Israel to 37% Lebanon). Additionally, between 21% (Japan) and 87% (Brazil) of smokers and ex-smokers had bought cigarettes when they knew the money could be spent better on household essentials like food.\n\nData are presented as unweighted n and weighted percentages\n\nBR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States\n\nTable 4 shows smokers’ quitting attempts and intentions to quit. In most countries a majority of smokers said they had tried to quit at least once, with more than half reporting multiple attempts. The range was similar when smokers were asked if they planned to quit, with 78% of smokers in New Zealand responding “yes” compared to 25% in Lebanon. Many of those planning to quit, however, indicated it would be in the future, beyond six months. Of those who were not planning to quit, up to half had previously attempted to quit.\n\nData are presented as unweighted n and weighted percentages\n\nBR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States\n\nSurvey participants listed concern about personal health the most often as a reason for quitting smoking, except in New Zealand and the UK where more smokers cited the price of tobacco products (Figure 6 and Figure 7). In addition to price, pressure from family, partner, and/or friends was another leading driver for both smokers and ex-smokers. There were some differences in motivation between smokers and ex-smokers, notably current Russian smokers find that smoking is becoming less fashionable, whereas ex-smokers’ concern for the impact of secondhand smoke made the top three factors for quitting in Japan and Lebanon. The impact of warning labels was among the top three reasons for quitting in India, Malawi, and Russia.\n\nWhen further questioned about the impact of price, a majority of smokers in 10 of the 13 countries surveyed said they would stop smoking, reduce their tobacco consumption, or switch to alternative products if the price of tobacco increased (Figure 8).\n\n% - top three answers. Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nData are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nSmokers: would an increase in tobacco price have an effect on your current smoking habit?. Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States. Column percentages may not add up to 100% due to rounding.\n\nFigure 9 highlights the main methods used by smokers and ex-smokers to quit or try to quit. The majority used no type of assistance. A minority of participants in all countries reported receiving support from a healthcare professional, other specialist, or from a specialized stop-smoking clinic when trying to quit.\n\nEx-smokers, or smokers who have tried to quit. Data are presented as unweighted n and weighted percentages. FR=France, UK=United Kingdom, US=United States.\n\nIn most countries, more than half of the smokers who had previously tried to quit and failed reported they would need assistance to quit (Figure 10). Among ex-smokers, up to 40% needed three or more attempts to quit successfully (Figure 11).\n\nSmokers: Let's imagine that you have to give up smoking completely tomorrow. Which of the following statements would best apply to you? (% - would need to seek assistance). Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nEx-smokers: How many times did you try to quit smoking before you were successful? Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States. Column percentages may not add up to 100% due to rounding.\n\nSmokers in almost all countries surveyed were less likely to describe their health as “excellent” or “very good” compared to never smokers (Table 5). More smokers in every country reported that they drank “too much” alcohol, and in most countries they reported feeling stressed in their personal or work lives more than never smokers (Table 5). Results on other variables such as weight, physical activity, healthy food consumption, and environmental factors were mixed between smokers and never smokers across countries (not shown).\n\nData are presented as unweighted n and weighted percentages\n\nResults are based on two-sided tests with significance level 0.05, Z ≥ 1.96 or Z ≤ −1.96\n\nBR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States\n\nParticipants stated they were well informed (very well informed or rather well informed) about the impact of smoking on their health (67% Malawi to 96% US) (Table 6). Further, they agreed (totally agree or tend to agree) that smoking was harmful to their own health (69% India to 96% Brazil) and to the health of others (66% India to 95% Greece). A majority of smokers were able to identify multiple conditions associated with smoking such as lung cancer and heart disease (not shown).\n\nData are presented as unweighted n and weighted percentages\n\nBR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States\n\nWhen asked to rate the harmfulness of cigarettes and other products such as wine, soda drinks, candy, junk food, and salty appetizers on one’s health, smokers as a group in all countries gave cigarettes the highest average harm rating compared to other products (Figure 12). When asked to rate the harmfulness of moderate daily use of the following substances: alcohol, caffeine, fat, nicotine, salt, and sugar, nicotine was given the highest average harm rating (Figure 13).\n\nSmokers: On a scale from 1 to 10, where 1 means not harmful to your health and 10 means very harmful to your health, to what extent do you think a moderate daily use of the following products can harm your health? (Ten point scale average: top three answers per country). Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nSmokers: On a scale from 1 to 10, where 1 means not harmful to your health and 10 means very harmful to your health, to what extent do you think a moderate daily use of the following substances can harm your health? (Ten point scale average: top three answers per country). Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States.\n\nMost participants in high income countries had heard of electronic cigarettes, e-cigarettes or vaping devices (ENDS, including devices with or without nicotine) (Figure 14). Awareness of heat-not-burn/heated tobacco products (HTP) among smokers was relatively low in all countries aside from Japan (86%) and Israel (52%).\n\nHave you heard of the following products? Electronic cigarettes, e-cigarettes or vaping devices. % - yes. Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States. ENDS=Electronic Nicotine Delivery Systems.\n\nThe perception by smokers regarding the relative harmfulness of ENDS compared with regular cigarettes was mixed, with many choosing not to answer, and the majority of smokers in only four countries believing that ENDS were less harmful than regular cigarettes (Figure 15). Data are also available regarding perceptions of relative harm to others through second hand smoke and vapor; of relative addictiveness to ENDS; and of association of nicotine in ENDS with various health conditions.\n\nSmokers: Do you think smoking e-cigarettes and vaping devices is more or less harmful than smoking regular cigarettes? Data are presented as unweighted n and weighted percentages. BR=Brazil, FR=France, GR=Greece, IL=Israel, IN=India, JP=Japan, LB=Lebanon, MW=Malawi, NZ=New Zealand, RU=Russia, SA=South Africa, UK=United Kingdom, US=United States. ENDS=Electronic Nicotine Delivery Systems.\n\nAnalysis of ENDS users was limited to France, UK, and US since the number of self-identified regular users of ENDS was too low in the remaining countries. The most often cited reasons to use ENDS were for decreasing or quitting smoking, however, among the top 3 reasons in the US and France was also the use of ENDS for enjoyment (Figure 16). More than half of these participants reported their tobacco consumption had decreased since regular use of ENDS (Figure 17). Additionally, one-fifth of these regular users indicated they choose products that do not contain nicotine (France 23.4%, UK 22%, US 20%).\n\nRegular users of ENDS. % - top 3 answers per country. Data are presented as unweighted n and weighted percentages. FR=France, UK=United Kingdom, US=United States. ENDS=Electronic Nicotine Delivery Systems.\n\nSince you started using these products, would say your tobacco consumption has increased, decreased or stayed the same? Data are presented as unweighted n and weighted percentages. Column percentages may not add up to 100% due to rounding. FR=France, UK=United Kingdom, US=United States. ENDS=electronic nicotine delivery systems.\n\n\nDiscussion\n\nThe FSFW global survey of smoking behavior and perceptions of more than 17,000 people in 13 countries identifies issues that will guide future efforts to stop smoking worldwide.\n\nThe results are consistent with other findings that show more male smokers, with women catching up in some countries and age groups4. We report a predominance of boxed cigarette use, while RYO is the second most common tobacco product in almost all countries, used by 2%–56% of smokers. The lower price of RYO compared to boxed cigarettes is cited by smokers and supported by price analyses as a reason for RYO use11,12. Our results show the highest number of RYO users being in NZ and UK, the two countries with the most expensive boxed cigarettes in this survey and which also had the most participants citing cost as a consideration for quitting smoking13.\n\nTaxing tobacco products is considered a best practice under the World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC)14 with global data demonstrating that increasing taxes on tobacco products is the most effective approach to reducing their use and encouraging users to quit1. A majority of smokers in this survey indicated they would change their smoking habits (stop smoking, reduce their tobacco consumption, or switch to alternative products) if the price of tobacco increased.\n\nThere is a concern that policies intended to incentivize smokers to quit are instead moving smokers away from highly taxed boxed cigarettes to a potentially more harmful alternative. RYO cigarettes vary in composition but have been shown to have higher tar yields than boxed cigarettes, as well as comparable exposure to known and suspected carcinogens15. Nevertheless, there is an erroneous belief among many users that RYO are a healthier alternative to boxed cigarettes11. An association of RYO users with lower educational or socioeconomic status, as well as a history of less stringent warning labels, may contribute to RYO users being less well informed of RYO tobacco risks11. More work is needed to address the continued rise of RYO use across countries.\n\nA majority of smokers in this survey generally characterize themselves as light or moderate smokers and most smoked daily. Nearly two-thirds or more of smokers considered themselves addicted to cigarettes. Across all countries, smokers associated smoking with daily routines. Smokers are surrounded by other smokers, and their smoking is tied to socially relevant activities such as meals, or drinking, whether it be coffee, tea, or alcohol.\n\nThe survey results indicate in most countries two-thirds of smokers try to quit without assistance, similar to rates reported elsewhere16. Behavioral therapies (group17 and individual18) and social supports19 that would address the deeply ingrained daily routines and social interactions of smokers have been shown to increase quit rates, however in a comparison of 22 national guidelines on smoking cessation, the recommended content and delivery of these therapies varies widely20. Although many smokers eventually quit without assistance21, on any given quit attempt, the success without assistance for remaining abstinent for at least 6–12 months is about 3–5%22.\n\nUsing the assistance of a healthcare professional or specialized stop-smoking clinic or specialist increases the likelihood of short and long-term quitting primarily through the advice to use, or prescription of, cessation medications21,23. Nicotine replacement therapies (NRT) increase the rate of quitting by 50–60%24, however, given the low rates of abstinence alone, this translates to an absolute efficacy increase in most populations of about 3%. A national sample of US adult smokers found just 40% of current smokers had ever used NRT such as patches, gum, or other products approved for smoking cessation, even though nearly all knew about these products25. In this survey, fewer than 20% sought specialized assistance and only a third or fewer smokers had tried NRT or other medications when trying to quit.\n\nOf those that failed in previous quit attempts, 14% (South Africa) to 57% (New Zealand) indicated they are not interested in trying again, underscoring the need for better and more comprehensive smoking cessation information and programs to increase quit success rates at the outset. Understanding the profiles of successful ex-smokers and of current smokers interested in quitting, the prime motivators to quit, and who is amenable to assistance can improve policies and outreach efforts for smokers seeking to quit.\n\nSmokers are largely aware of the health consequences of smoking and it is the most often cited reason for quitting among ex-smokers, and for current smokers in most countries.\n\nHowever, while smokers are broadly correct in acknowledging the harm of cigarettes, many are confused as to the source of the harm. When asked to rate the harmfulness of cigarettes, most rate cigarettes as more harmful than other products such as wine, junk food, soda, salty appetizers, and candy. However, when asked to rate the harmfulness of moderate daily use of nicotine to their own health, smokers again rate nicotine very high, exceeding or matching every other substance (salt, fat, sugar, alcohol, caffeine). In a 2015 national US survey, nearly one-half believed nicotine in cigarettes is the main cause of smoking related cancer, and another 24% were unsure26. Other surveys report similar misconceptions about nicotine in NRT, with 21% of smokers believing the patch is associated with heart problems25, and two thirds of a pool of smokers and ex-smokers agreeing or unsure that “stop-smoking products with nicotine are just as harmful as cigarettes”27. Further research to parse out participants’ intentions in rating harmfulness of nicotine is needed, as well as asking about tobacco as a substance for comparison. Misperceptions of the role of nicotine could be limiting public health efforts to curtail smoking, including contributing to low uptake of NRT28 or confusion regarding reduced-risk products26.\n\nTobacco control includes substitution of higher harm products with lower harm products14. While the consensus holds ENDS are substantially less harmful than traditional cigarettes29,30, public health messaging regarding use varies. In the UK, ENDS are promoted for smoking cessation whereas the WHO recommends regulatory measures to protect against possible health risks28. Some countries, including several in this study, have restricted or banned sale and/or possession of ENDS31. These competing messages regarding ENDS appear to add to the misunderstanding of the role of nicotine32. In two-thirds of surveyed countries there was a very high level of awareness of ENDS, however, many smokers were unable or unwilling to categorize whether ENDS were more, less, or equally harmful to health compared with regular cigarettes.\n\nENDS users in this survey most frequently cite adopting ENDS to quit or cut down on smoking. In a longitudinal survey of US adult smokers, substituting ENDS for some cigarettes when trying to quit was a method used more often than the nicotine patch or gum, or other smoking cessation medications approved by the US Food and Drug Administration, with one-quarter of the most recent quit attempts replacing all cigarettes with ENDS33. Compared to other nicotine, non-tobacco products, ENDS most closely simulate smoking regular cigarettes in how they are used. The variety of products allow users to customize their experience in terms of flavor and amount of nicotine which could further enhance ENDS as a replacement for traditional cigarettes.\n\nA majority of ENDS users in countries with sufficient sample size in this survey report decreased tobacco consumption since starting ENDS. Studies to date tend to focus on ENDS use and quit rates as absolutes rather than assessing the relative harm reduction, especially when considering the risk status of a smoker versus a never smoker. Models that consider a public health perspective of ENDS are positive, with 1.6 million or more fewer premature deaths over 10 years, even in scenarios where not all smokers quit when using ENDS, some never smokers become ENDS users, and more harm is attributed to ENDS than has been currently found34. Further investigation and research of ENDS use and other alternatives along this spectrum of harm reduction is needed.\n\nThis survey is limited by potential response bias in those choosing to participate, and reporting bias as there were no external or objective validations. Although the survey results were weighted according to population figures, the sampling was not strictly designed to estimate overall smoking prevalence. Additionally, while the countries included in the survey were chosen to represent a range of income levels and smoking prevalence, generalizability to other countries may be limited by cultural norms and regulations. There may have been differences between surveys administered face-to-face versus online. Despite attention to using previous surveys as a guide to create and pilot the survey, several deficiencies emerged such as quantifying the number of cigarettes smoked. Future surveys will seek to include more questions on the understanding of the role of nicotine, as well as comparisons to other substances, including tobacco. Questions regarding the use of ENDS will eliminate the word “smoking” to reduce confusion regarding the use of tobacco versus non-tobacco products and include more detailed categories for frequency of use.\n\n\nConclusion\n\nThis global survey highlights several areas of global smoking behavior and perceptions that need particular attention, namely the deeply social and behavioral aspects of smoking, the inadequacy of current efforts to promote quitting, the role of RYO cigarettes, and the confusion many smokers have regarding tobacco and nicotine products, including ENDS.\n\nThis survey report represents the initial piece of FSFW’s work to improve global health and end smoking in this generation. Next steps include a more detailed look at the economic levers of tobacco preference, the perceived harms of cigarette smoking vs the objective realities of these harms, and a look at the characteristics and choices of ex-smokers.\n\nFSFW is committed to funding research, promoting innovation, and supporting collaborative initiatives to accelerate progress in reducing harm and death from smoking worldwide. To this end, data from this survey are available online for further analyses, and FSFW welcomes input for follow-up surveys.\n\n\nData availability\n\nOpen Science Framework: Global Poll 2018, https://doi.org/10.17605/OSF.IO/X4GQZ35\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nKantar Public methodology report and 81-question quantitative survey: https://amadashboards.com/kp/eu_smokefree/Doc/FSFW%20-%20State%20of%20Smoking%20Survey%20-%20Method%20statement.pdf", "appendix": "Grant information\n\nThis survey was fully funded by FSFW, and no grants or other financial sources were involved.\n\n\nAcknowledgements\n\nThe authors would like to thank Francine Wiest, independent researcher, for writing support, Madeleine Smith for data analysis support, and Olivier Parnet, Director of Opinion & Impact, for assistance with data management and analyses.\n\n\nReferences\n\nWorld Health Organization: WHO report on the global tobacco epidemic, 2017: Monitoring tobacco use and prevention policies. Accessed Dec 5, 2017. Reference Source\n\nInstitute for Health Metrics and Evaluation: Daily smoking patterns for both sexes, all ages. 2017; Accessed Aug 20, 2018. Reference Source\n\nNg M, Freeman MK, Fleming TD, et al.: Smoking Prevalence and Cigarette Consumption in 187 Countries, 1980-2012. JAMA. 2014; 311(2): 183–192. PubMed Abstract \| Publisher Full Text\n\nGBD 2015 Tobacco Collaborators: Smoking prevalence and attributable disease burden in 195 countries and territories, 1990-2015: a systematic analysis from the Global Burden of Disease Study 2015. Lancet. 2017; 389(10082): 1885–1906. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrope J, Schluger N, Cahn Z, et al.: The Tobacco Atlas. 6th edition. Atlanta: American Cancer Society and Vital Strategies. 2018; Accessed Dec 5, 2018. Reference Source\n\nBabb S, Malarcher A, Schauer G, et al.: Quitting Smoking Among Adults - United States, 2000-2015. MMWR Morb Mortal Wkly Rep. 2017; 65(52): 1457–64. PubMed Abstract \| Publisher Full Text\n\nEuropean Commission: Attitudes of Europeans Towards Tobacco and Electronic Cigarettes. 2015. Reference Source.\n\nYach D: Foundation for a smoke-free world. Lancet. 2017; 390(10104): 1807–10. PubMed Abstract \| Publisher Full Text\n\nGottlieb S, Zeller M: A Nicotine-Focused Framework for Public Health. N Engl J Med. 2017; 377(12): 1111–4. PubMed Abstract \| Publisher Full Text\n\nAbrams DB, Glasser AM, Pearson JL, et al.: Harm Minimization and Tobacco Control: Reframing Societal Views of Nicotine Use to Rapidly Save Lives. Annu Rev Public Health. 2018; 39: 193–213. PubMed Abstract \| Publisher Full Text\n\nBrown AK, Nagelhout GE, van den Putte B, et al.: Trends and socioeconomic differences in roll-your-own tobacco use: findings from the ITC Europe Surveys. Tob Control. 2015; 24(Suppl 3): iii11–iii16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRothwell L, Britton J, Bogdanovica I: The relation between cigarette price and hand-rolling tobacco consumption in the UK: an ecological study. BMJ Open. 2015; 5(6): e007697. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrice Rankings by Country of Cigarettes 20 Pack (Marlboro) Markets. Accessed Dec 5, 2018. Reference Source\n\nWorld Health Organization: Framework Convention on Tobacco Control. Accessed Oct 18 2018. Reference Source\n\nFowles JL: Mainstream smoke emissions from 'RYO' loose-leaf tobacco sold in New Zealand Report to the New Zealand Ministry of Health. 2008; Accessed Dec 5, 2018. Reference Source\n\nSoulakova JN, Crocket LJ: Level of Cigarette Consumption and Duration of Smoking Abstinence During Failed Quit Attempts Among Long-Term Daily Smokers: the Role of Race/Ethnicity and Cessation Aids. J Racial Ethnic Health Disp. 2018; 5(2): 293–303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStead LF, Carroll AJ, Lancaster T: Group behaviour therapy programmes for smoking cessation. Cochrane Database Syst Rev. 2017; 3: CD001007. PubMed Abstract \| Publisher Full Text\n\nLancaster T, Stead LF: Individual behavioural counselling for smoking cessation. Cochrane Database Syst Rev. 2017; 3: CD001292. PubMed Abstract \| Publisher Full Text\n\nU.S. Department of Health and Human Services, Public Health Service: Treating Tobacco Use and Dependence: 2008 Update. Content last reviewed October 2018. Agency for Healthcare Research and Quality, Rockville, MD. Accessed Dec 5, 2018. Reference Source\n\nVerbiest M, Brakema E, van der Kleij R, et al.: National guidelines for smoking cessation in primary care: a literature review and evidence analysis. NPJ Prim Care Respir Med. 2017; 27(1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoulakova JN, Crockett LJ: Unassisted Quitting and Smoking Cessation Methods Used in the United States: Analyses of 2010-2011 Tobacco Use Supplement to the Current Population Survey Data. Nicotine Tob Res. 2017; 20(1): 30–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes JR, Keely J, Naud S: Shape of the relapse curve and long-term abstinence among untreated smokers. Addiction. 2004; 99(1): 29–38. PubMed Abstract \| Publisher Full Text\n\nZhang B, Chaiton MO, Diemert LM, et al.: Health professional advice, use of medications and smoking cessation: A population-based prospective cohort study. Prev Med. 2016; 91: 117–22. PubMed Abstract \| Publisher Full Text\n\nHartmann-Boyce J, Chepkin SC, Ye W, et al.: Nicotine replacement therapy versus control for smoking cessation. Cochrane Database Syst Rev. 2018; 5: CD000146. PubMed Abstract \| Publisher Full Text\n\nBansal MA, Cummings KM, Hyland A, et al.: Stop-smoking medications: who uses them, who misuses them, and who is misinformed about them? Nicotine Tob Res. 2004; 6 Suppl 3: S303–10. PubMed Abstract \| Publisher Full Text\n\nO'Brien EK, Nguyen AB, Persoskie A, et al.: U.S. adults' addiction and harm beliefs about nicotine and low nicotine cigarettes. Prev Med. 2016; 96: 94–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShiffman S, Ferguson SG, Rohay J, et al.: Perceived safety and efficacy of nicotine replacement therapies among US smokers and ex-smokers: relationship with use and compliance. Addiction. 2008; 103(8): 1371–8. PubMed Abstract \| Publisher Full Text\n\nConference of the Parties to the WHO Framework Convention on Tobacco Control; FCTC/COP6(9) Decision: Electronic nicotine delivery systems and electronic non-nicotine delivery systems. World Health Organization. 2014; Accessed Dec 5, 2018. Reference Source\n\nNational Academies of Sciences, Engineering, and Medicine: Public health consequences of e-cigarettes. Washington, DC: The National Academies Press. 2018. Publisher Full Text\n\nMcNeill A, Brose LS, Calder R, et al.: Evidence review of e-cigarettes and heated tobacco products 2018. London: Public Health England. 2018; Accessed Dec 5, 2018. Reference Source\n\nKennedy RD, Awopegba A, De León E, et al.: Global approaches to regulating electronic cigarettes. Tob Control. 2017; 26(4): 440–445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAction on Smoking and Health: Use of e-cigarettes (vapourisers) among adults in Great Britain. 2018; Accessed Dec 5, 2018. Reference Source\n\nCaraballo RS, Shafer PR, Patel D, et al.: Quit Methods Used by US Adult Cigarette Smokers, 2014–2016. Prev Chronic Dis. 2017; 14: E32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevy DT, Borland R, Lindblom EN, et al.: Potential deaths averted in USA by replacing cigarettes with e-cigarettes. Tob Control. 2018; 27(1): 18–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajkumar S: Global Poll 2018. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/X4GQZ" }	[ { "id": "44210", "date": "26 Feb 2019", "name": "Kenneth Michael Cummings", "expertise": [ "Reviewer Expertise Tobacco control and behavioral 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 is a nicely written paper with lots of great data. However, the paper can be improved upon by providing information on statistical weighting. The description of how weighting of data from what appears to be convenience samples done in different countries is inadequate. In order to generate samples that are weighted so as to reflect the country specific estimates that are provided requires having some country specific source data for each of the 13 countries for which there are survey estimates presented. The question is what source data were used for each country and how closely do the characteristics of the samples from each country match the gold standard source dataset used to generate sampling weights? The authors may have a technical report that investigates this issue and can provide the evidence needed so that readers can feel confident in the country specific estimates provided. However, the paper seems to lack this information, which is critical.\nI was concerned that the investigators did not seek human subjects review for their surveys. I agree the surveys are low risk. However, I think this is something that the investigators ought to address in subsequent survey work that they might undertake. Many journals will not accept such findings without such certification. It was unclear from the methods write up if subjects were compensated for their time taking the survey which is normally done. Was that the case in the survey described and how did compensation differ between countries?\nCountry names should either be spelled out or abbreviations, if used described at least one time in the text (i.e., FR=France, BR=Brazil, etc. - see table 1). The paper should include definitions of products as it is likely that in Indian bidis are being referred to as cigarettes.\n\nAlso, India is a big country, what regions of the country were surveyed, and how does region impact product use (i.e., higher in some places for smokeless vs bidis). How is perceived addiction correlated with regular daily use?\nIn Table 3 it appears that those from poor countries are more likely to report using tobacco instead of food. That should be stated. In Table 4, add the questions on have tried to quit once with have tried to quit more than once to create an overall percentage who have reported having tried to quit.\nOn page 10, the statement that RYO tobacco yields higher tar levels compared to boxed tobacco is untrue and misleading. RYO tobacco is most often commercial tobacco packaged for rolling. RYO tobacco packaged in this way typically has more additives (usually for moisture). Tar and nicotine content is mostly determined by the amount rolled which is highly variable. The one reference cited is old, out of date, and perhaps correct for New Zealand at the time, but not generally true world wide. The industry loves to say RYO tobacco is worse than boxed tobacco and that is simply false. Also on page 10, RYO is widely used form of tobacco in many countries - this should be reported.\n\nThe comment on page 12 assumes health professional advice would be expected to be the same across countries. That is simply not realistic. Thus such comparisons are misleading (See paper by Borland et al, One size does not fit all when it comes to smoking cessation: observations from the International Tobacco Control (ITC) Policy Evaluation Project)1. Table 5 is unreadable.\nOn page 17, modelling of ENDS effects on health are only really focused on the US and other high income countries. It is unlikely that ENDS would have much impact in countries with very low smoking rates. Also the term ENDS is out of date and ought to be replaced with a broader term - nicotine vaping products (NVPs).\nThe data from the study ought to be made publicly available. While lots of interesting descriptive data are provide, the paper loses its value since the authors have not focused the presentation on any themes. I would advise reformatting the paper, and use a fewer number of tables to communicate a few key themes. Additional materials can be placed in supplemental tables/figures. I would also encourage the authors to use figures where possible since this would be easier for readers to follow.\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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4611", "date": "13 May 2019", "name": "Sarah Rajkumar", "role": "Author Response", "response": "This is a nicely written paper with lots of great data. However, the paper can be improved upon by providing information on statistical weighting. The description of how weighting of data from what appears to be convenience samples done in different countries is inadequate. In order to generate samples that are weighted so as to reflect the country specific estimates that are provided requires having some country specific source data for each of the 13 countries for which there are survey estimates presented. The question is what source data were used for each country and how closely do the characteristics of the samples from each country match the gold standard source dataset used to generate sampling weights? The authors may have a technical report that investigates this issue and can provide the evidence needed so that readers can feel confident in the country specific estimates provided. However, the paper seems to lack this information, which is critical.Response: The methods statement that is available on our website provides more detailed tables on the weighting estimates, starting on page 16. We provide a link to it at the end of the manuscript. The population distribution was taken from the most recent national census data available. We have added this to the paper as well when describing the weighting procedure.I was concerned that the investigators did not seek human subjects review for their surveys. I agree the surveys are low risk. However, I think this is something that the investigators ought to address in subsequent survey work that they might undertake. Many journals will not accept such findings without such certification. It was unclear from the methods write up if subjects were compensated for their time taking the survey which is normally done. Was that the case in the survey described and how did compensation differ between countries?Response: We thank the reviewer for his recommendation to seek IRB approval in our next survey as indeed omitting this could lead to an article being rejected right away.Face-to-face respondents were not compensated for their participation. Online respondents were all members of an online panel company. As such, according to the industry standard, they received Reward Points, to encourage panelists to participate in surveys. Upon completion of a survey, points are deposited into a panelist’s account, which gives instant gratification for survey completion. The number of points awarded for survey completion is based on survey length, complexity, and incidence rate. Once a points threshold is reached, panelists may redeem their points for online gift certificates or merchandise. Each country has its own unique catalogue. We have added this information to the methods section on page 4.Country names should either be spelled out or abbreviations, if used described at least one time in the text (i.e., FR=France, BR=Brazil, etc. - see table 1).Response: We have ensured that all abbreviations are spelled out at first use.The paper should include definitions of products as it is likely that in Indian bidis are being referred to as cigarettes. Response: We have added bidis to the description of products that smokers use as indeed they were included for the Indian population.Also, India is a big country, what regions of the country were surveyed, and how does region impact product use (i.e., higher in some places for smokeless vs bidis).Response: India was divided into four geographical regions - i.e. North, South, East and West and results were weighted according to the population distribution. One state was randomly sampled from each geographic region. The selected states were: Uttar Pradesh (North), Telangana (South), West Bengal (East) and Gujarat (West). We agree that India is too large and diverse a country to integrate all numbers into one result but our sample size was too small to report meaningful data on a sub-national level. We are amending this in our next poll which will be carried out in 2019 with a much larger sample size in India.How is perceived addiction correlated with regular daily use?Response: We have decided to only report descriptive numbers and no correlation numbers or further statistical analyses. While exploring correlations between some of the outcomes would certainly be interesting, adding correlations here would go beyond the scope of the present paper.In Table 3 it appears that those from poor countries are more likely to report using tobacco instead of food. That should be stated.Response: We have added this statement to the paragraph where we describe results from Table 3.In Table 4, add the questions on have tried to quit once with have tried to quit more than once to create an overall percentage who have reported having tried to quit.Response: Done.On page 10, the statement that RYO tobacco yields higher tar levels compared to boxed tobacco is untrue and misleading. RYO tobacco is most often commercial tobacco packaged for rolling. RYO tobacco packaged in this way typically has more additives (usually for moisture). Tar and nicotine content is mostly determined by the amount rolled which is highly variable. The one reference cited is old, out of date, and perhaps correct for New Zealand at the time, but not generally true world wide. The industry loves to say RYO tobacco is worse than boxed tobacco and that is simply false.Response: We have deleted this statement.Also on page 10, RYO is widely used form of tobacco in many countries - this should be reported.Response: We report this at the beginning of the results section and the discussion.The comment on page 12 assumes health professional advice would be expected to be the same across countries. That is simply not realistic. Thus such comparisons are misleading (See paper by Borland et al, One size does not fit all when it comes to smoking cessation: observations from the International Tobacco Control (ITC) Policy Evaluation Project)1.Response: We mention on page 12 that “in a comparison of 22 national guidelines on smoking cessation, the recommended content and delivery of these therapies varies widely”. We acknowledge that this is a limitation in a survey across so diverse a selection of countries.Table 5 is unreadable.Response: Please refer to the pdf version of the paper as this table has been distorted in the edited word document.On page 17, modelling of ENDS effects on health are only really focused on the US and other high income countries. It is unlikely that ENDS would have much impact in countries with very low smoking rates. Also the term ENDS is out of date and ought to be replaced with a broader term - nicotine vaping products (NVPs).Response: We only included France, the UK and the US in our reporting of ENDS use as dispersion of ENDS in the other countries is currently too low to yield any meaningful numbers given our sample sizes.We strongly agree that there is a need for harmonization regarding the terminology, nevertheless we suggest sticking to ENDS in this paper as this was term used in the survey.The data from the study ought to be made publicly available.Response: Data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).Open Science Framework: Global Poll 2018, https://doi.org/10.17605/OSF.IO/X4GQZThese links are available at the end of the paper.While lots of interesting descriptive data are provide, the paper loses its value since the authors have not focused the presentation on any themes. I would advise reformatting the paper, and use a fewer number of tables to communicate a few key themes. Additional materials can be placed in supplemental tables/figures. I would also encourage the authors to use figures where possible since this would be easier for readers to follow.Response: The objective of this paper was to give a very broad overview of quantitative results from the first Global Poll commissioned by the foundation. Therefore, we provided only descriptive analysis and did not perform any in-depth analysis by ways of more complex statistical models. We have decided to narrow down our focus in our next poll and are planning to publish more specific results from there.We thank the reviewer for taking the time to thoroughly review this paper." } ] }, { "id": "46336", "date": "01 Apr 2019", "name": "Axel Klein", "expertise": [ "Reviewer Expertise qualitative and anthropological research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well designed and exhaustive study of attitudes among smoking/vaping populations and explores some of the obstacles to changing behaviour among different cohorts of smokers in different countries. There are interesting variations between for instance Lebanon where high levels of smoking continue to suggest a 'normalised' view of smoking particularly among adult males and countries where smoking is waning and, as the Russian informants reports, no longer fashionable.\n\nThis formulation is important as it suggests that there are population groups that are not influenced by any of the established means of suasion - information provision on negative health effect, appeal and reconstruction of self image. It undermines both the conceptual model of homo oeconomicus, the rational, means/ends calculating rational analyst or the affective, fashion oriented, decision maker.\n\nInteresting data is also coming up about the attitudes to ENDS as an alternative delivery vehicle for tobacco. First, there is a deep rooted misunderstanding of adverse health effects of smoking. Large sections of the population (including physicians) continue to believe that it is the nicotine content of tobacco that is carcinogenic. It is this misconception that may set a block to transitioning to vaping mechanisms as people may think that vaping is just as dangerous because nicotine is not excluded.\nThere emerges then a strong policy implication. Public health agencies must become more assertive in pressing the case for vaping. Moreover, the campaign by some lobbies with a view to stigmatize all nicotine use behaviour is not only discriminatory, but also highly irresponsible in public health terms by raising resistance to behaviour change.\n\nThrough the sheer scale of the research large areas in the smoking cessation/transition debate are well illustrated. In many ways this is a starting point for correcting policy and practice measures and for driving new qualitative research to get a better understanding of the human beings behind the labels of smoker/vaper/nicotine users/ex-smoker and so on. It is also a good platform from where to explore and construe the narratives of transition, redefinitions, new-nicotine sociality and the search for a renewed definition of pleasure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-80

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